Bcl-g polypeptides, encoding nucleic acids and methods of use

ABSTRACT

The invention provides Bcl-G polypeptides and encoding nucleic acids. Bcl-G polypeptides include Bcl-G L  and Bcl-G S . The invention also provides mouse Bcl-G. The invention also provides vectors containing Bcl-G nucleic acids, host cells containing such vectors, Bcl-G anti-sense nucleic acids and related compositions. The invention additionally provides Bcl-G oligonucleotides that can be used to hybridize to or amplify a Bcl-G nucleic acid. Anti-Bcl-G specific antibodies are also provided. Further provided are kits containing Bcl-G nucleic acids or Bcl-G specific antibodies. Such kits and reagents can be used to diagnose cancer, monitor response to therapy, or predict the prognosis of a cancer patient. The invention additionally provides methods of modulating apoptosis using Bcl-G polypeptides, encoding nucleic acids, or compounds that modulate the activity or expression of Bcl-G polypeptides. The methods for modulating apoptosis can be used to treat diseases such as cancer.

This application is a continuation of application Ser. No. 12/646,747,filed Dec. 23, 2009, which is a divisional of application Ser. No.09/738,396, filed Dec. 14, 2000, now U.S. Pat. No. 7,638,324, whichclaims the benefit of U.S. Provisional Application No. 60/287,581, filedDec. 14, 1999, which was converted from U.S. Ser. No. 09/461,641, filedDec. 14, 1999, each of which the entire contents are incorporated hereinby reference.

This invention was made with government support under grant numberGM60554 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to regulation of programmed celldeath and more specifically to molecules that promote programmed celldeath.

In essentially all self-renewing tissues, a balance is struck betweencell production by mitogenesis and cell loss due to programmed celldeath, thereby maintaining total cell numbers within a physiologicallyappropriate range. In pathological conditions, however, the balance incell production and cell loss can be disrupted. In cancer, for example,an increased amount of cell production due to a shortened cell cycletime or a decreased amount of cell death due to dysregulation of aprogrammed cell death pathway results in the growth of a tumor.

With regard to programmed cell death, a variety of stimuli, which occureither external or internal to the cell, initiate a pathway thatultimately results in apoptosis of the cell. As is common for mostsignal transduction pathways, the various different stimuli that induceapoptosis likely initiate the process of programmed cell death throughspecific pathways. However, most if not all of these initial pathwaysconverge at a common point that generally involves a member of the Bcl-2family of proteins.

The Bcl-2 family of proteins regulate a distal step in theevolutionarily conserved pathway for programmed cell death andapoptosis, with some members of this family functioning as suppressorsof cell death (anti-apoptotic proteins) and other members functioning aspromoters of cell death (pro-apoptotic proteins). Overexpression of theanti-apoptotic protein, Bcl-2, for example, blocks neuronal cell deaththat otherwise is induced in vitro by various stimuli, includingneurotrophic factor withdrawal, various oxidants, glucose deprivation,certain neurotrophic viruses, and amyloid β-peptide. In addition, Bcl-2is overexpressed in some tumor cells and, in part, may contribute totumor growth by altering the balance between cell division and celldeath.

The Bcl-2 family of proteins are critical regulators of pathwaysinvolved in apoptosis, acting to either inhibit or promote cell death(Reed, Nature 387:773-776 (1997); Green and Reed, Science 281:1309-1312(1998); Reed, Oncogene 17:3225-3236 (1998); Reed, Curr. Opin. Oncol.11:68-75 (1999)). The Bcl-2 family members can be divided into twogroups, those with anti-apoptotic activity, including Bcl-2 andBcl-X_(L), and those with pro-apoptotic activity, including Bax and Bak.

Four distinct domains have been identified in Bcl-2 family members,designated BH1 to BH4. The BH4 domain is a domain that mediatesinteractions with a variety of cellular proteins (Reed, supra, 1998).The BH1, BH2 and BH3 domains form a binding pocket for dimerization withother Bcl-2 members having a BH3 domain, which also functions as aligand that binds to the dimerization binding pocket. The dimerizationfunction of the Bcl-2 members is an important mechanism for regulatingapoptosis in that heterodimerization of pro-apoptotic Bcl-2 members withanti-apoptotic Bcl-2 members can regulate the cellular apoptoticpathways. Some Bcl-2 members only have a BH3 domain and thereforefunction as trans-dominant inhibitors of anti-apopototic proteins suchas Bcl-2 and Bcl-X_(L) (Reed, supra, 1998).

Another function of Bcl-2 members is the formation of ion channels.Bcl-2 members can localize to the mitochondrial membrane, and theformation of ion pores that alter the permeability of mitochondria isthought to be an important signaling mechanism for the induction ofapoptosis. Thus, Bcl-2 members use at least three mechanisms to regulateapoptotic activity: dimerization with Bcl-2 members, formation of ionpores in mitochondria, and binding to non-Bcl-2 members that function assignaling molecules.

In comparison, overexpression of the pro-apoptotic protein, Bax, forexample, promotes cell death when triggered by a variety of inducers ofapoptosis, including growth factor withdrawal, ionizing radiation, andanti-Fas antibody. In addition, elevations in Bax expression occur inassociation with cell death induced by a variety of stimuli, includingneuronal cell death that occurs due to ischemia, epilepsy, spinal cordinjury, and certain neurodegenerative diseases such as Parkinson'sdisease and Alzheimer's disease.

Although aberrant expression of members of the Bcl-2 family of proteinsis associated with various pathologic conditions, the mechanisms bywhich these proteins mediate their action is not known. Often, theaction of a protein can be inferred from its structural relationship toother proteins, whose functions are known. However, while the Bcl-2family proteins share certain structural homologies with each other,they do not share substantial amino acid sequence homology with otherproteins, further hindering attempts to understand how the Bcl-2 familyproteins such as Bcl-2 and Bax regulate cell death.

Thus, a need exists to identify proteins involved in the programmed celldeath pathway and to identify methods of regulating programmed celldeath for therapeutic applications, including treatment of cancer. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are provided Bcl-Gpolypeptides and encoding nucleic acid molecules. The inventionpolypeptides and encoding nucleic acids are useful for modulatingapoptosis by altering the expression or activity of Bcl-G. The Bcl-Gpolypeptides and encoding nucleic acids can be advantageously used todiagnose or treat cancer, in particular prostate, ovarian and leukemia.Furthermore, the Bcl-G polypeptides and encoding nucleic acids areuseful to generate or screen for agents that can alter Bcl-G activity orexpression, which can further be used to treat cancer. Bcl-Gpolypeptides include Bcl-G_(L) and Bcl-G_(S).

The invention also provides vectors containing Bcl-G nucleic acids, hostcells containing such vectors, Bcl-G anti-sense nucleic acids andrelated compositions. The invention additionally provides Bcl-Goligonucleotides that can be used to hybridize to or amplify a Bcl-Gnucleic acid. Anti-Bcl-G specific antibodies are also provided. Furtherprovided are kits containing Bcl-G nucleic acids or Bcl-G specificantibodies. Such kits and reagents can be used to diagnose cancer,monitor response to therapy, or predict the prognosis of a cancerpatient. The invention additionally provides methods of modulatingapoptosis using Bcl-G polypeptides, encoding nucleic acids, or compoundsthat modulate the activity or expression of Bcl-G polypeptides. Themethods for modulating apoptosis can be used to treat diseases such ascancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of human Bcl-G_(L) cDNA (SEQ IDNO:1).

FIG. 2 shows the nucleotide sequence of the coding region of humanBcl-G_(L) cDNA (nucleotide 196-1179 of SEQ ID NO:1) and the encodedamino acid sequence (SEQ ID NO:2). Bcl-G_(L) contains a BH3 domain(²¹⁶LKYSGDQLE²²⁴; SEQ ID NO:5) and a BH2 domain (³⁰⁷PWIQQHGGWE³¹⁶; SEQID NO:6).

FIG. 3 shows the nucleotide sequence of human Bcl-G_(S) cDNA (SEQ IDNO:3).

FIG. 4 shows the nucleotide sequence of the coding region of humanBcl-G_(S) cDNA (nucleotide 196-954 of SEQ ID NO:3) and the encoded aminoacid sequence (SEQ ID NO:4). Bcl-G_(S) contains only the BH3 domain(²¹⁶LKYSGDQLE²²⁴; SEQ ID NO:5).

FIGS. 5A-5D show sequence analysis of Bcl-G cDNAs. FIG. 5A shows thepredicted amino acid sequences of the Bcl-G_(L) and Bcl-G_(S) proteins,with the BH2 and BH3 domains in bold-type and residue numbers indicated.The predicted proteins are identical from residues 1-226. The uniqueC-terminal region of Bcl-G_(S) is indicated in italics type. FIG. 5Bshows an alignment of the BH2 domains of Bcl-G_(L) (SEQ ID NO:9) andseveral other Bcl-2 family proteins (SEQ ID NOS:10-17, respectively).Identical and similar residues are shown in black and gray blocks,respectively. FIG. 5C shows an alignment of the BH3 domains Bcl-G (SEQID NO:18) and several other Bcl-2 family proteins (SEQ ID NOS:19-26,respectively). FIG. 5D shows the exon-intron organization of the BCL-Ggene. The BCL-G gene contains 6 exons, spanning a ˜30 kb region ofchromosome 12. Alternative splicing at the 5′-end of exon 5 accounts forthe production of the Bcl-G_(L) and Bcl-G_(S) proteins, wheresplice-acceptor sites at nucleotide positions 63,870 versus 63,797 inBAC clone RPCI 11-267J23 (GenBank AC007537) are utilized for Bcl-G_(L)and Bcl-G_(S), respectively. The positions of the start and terminationcodons are indicated, with coding regions in gray blocks and non-coding5′-UTR and 3′-UTR sequence in open blocks. The BH3 domain is located inexon 4 of both Bcl-G_(L) and Bcl-G_(S), while the BH2 domain resides inexon 5 of Bcl-G_(L).

FIG. 6 shows mapping of Bcl-G to chromosome 12p12.3.

FIGS. 7A and 7B show expression of Bcl-G_(S) and Bcl-G_(L) in humantissues. The expression of transcripts encoding Bcl-G_(L) or Bcl-G_(S)was examined by RT-PCR. First-strand cDNA prepared using RNA samplesfrom various adult human tissues was PCR amplified using primersspecific for Bcl-G_(L) and Bcl-G_(S), based on differences insplice-acceptor utilization in exon 5. The primers flank an intron inboth cases, thus excluding amplification due to contaminating genomicDNA. PCR products were size-fractionated in 2% agarose gels, stainedwith ethidium bromide, then photographed under UV-illumination.

FIG. 8 shows the effect of Bcl-G_(S) on cell death. PC-3 cells weretransfected with pcDNA3.1/Myc/His (control), pcDNA3.1/Myc/His/Bcl-G_(S)(Bcl-G_(S)), pcDNA3.1/Myc/His/Bcl-G_(S)+pRC/CMV/Bcl-2 (Bcl-G_(S)+Bcl-2),pRC/CMV/Bax (Bax), or pRC/CMV/Bax+pRC/CMV/Bcl-2 (Bax+Bcl-2). Cells weretested for cell death 24 hours after transfection.

FIGS. 9A and 9B show induction of apoptosis by Bcl-G. FIG. 9A shows theresults of transfecting plasmids encoding GFP, GFP-Bcl-G_(S), orGFP-Bcl-G_(L) into Cos-7 cells alone or in combination with a plasmidencoding Bcl-X_(L). Apoptosis was examined by DAPI staining at 24 hpost-transfection (mean±SD; n=3) (top). Levels of GFP and GFP-Bcl-Gfusion proteins were examined by immunoblotting lysates from transfectedCos-7 cells (20 μg per lane) and anti-GFP antibody with ECL-baseddetection (middle). Equal loading was confirmed by reprobing the samemembrane with anti-Tubulin antibody (bottom). FIG. 9B shows the resultsof transfecting plasmids encoding GFP, GFP-Bcl-G_(S) or the mutantproteins, Bcl-G_(S) (ΔBH3) and GFP-Bcl-G_(S) (L216E) into Cos-7 cells.The percentage of apoptotic cells was examined 1 day later as above(top). Protein expression was assessed by immunoblotting as above, usinganti-GFP (middle) or anti-Tubulin (bottom) antibodies.

FIGS. 10A-10C show interactions of Bcl-G_(S) and Bcl-G_(L) withBcl-X_(L). 293T cells were transiently transfected with plasmidsencoding GFP, GFP-Bcl-G_(L), GFP-Bcl-G_(S), GFP-Bcl-G_(S) (DBH3), orGFP-Bcl-G_(S) (L218E). Cells were lysed 1 day later andimmunoprecipitations were performed using anti-GFP antibody.Immune-complexes (prepared from 2 mg lysate) (top) and lysates (20 μgprotein) (bottom) were subjected to SDS-PAGE/immunoblot analysis usinganti-Bcl-X_(L) (top) and anti-GFP (bottom) antibodies, respectively.

FIGS. 11A-11D show microscopic evaluation of intracellular distributionsof Bcl-G_(L) and Bcl-Gs. Plasmids encoding GFP (FIG. 11A), GFP-Bcl-G_(L)(FIG. 11B), GFP-Bcl-G_(S) (FIG. 11C), and GFP-Bcl-G_(S) (ΔBH3) (FIG.11D) were transfected into Cos-7 cells. Cells were fixed 1 day later andexamined by confocal microscopy.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided nucleicacids encoding Bcl-G polypeptides, or functional polypeptide fragmentsthereof. As used herein, the term

Bcl-G@ refers to sub-family members of the Bcl-2 family of proteins,wherein said Bcl-G comprises a BH3 domain (SEQ ID NOS:5 or 9). The humanBcl-G gene has been found to map to chromosome 12p12.3 (Example II).This region of chromosome 12 is frequently deleted in cancer cells, inparticular in acute lymphoblastic leukemia (ALL) and other solid tumorcells (Baens et al., (1999) Genomics 56:40-50 (1999); Hatta et al., Br.J. Cancer 75:1256-1262 (1997); Kibel et al., Cancer Res. 58:5652-5655(1998); Baccichet et al., Br. J. Haematol. 99:107-114 (1997); Aissani etal., Leuk. Lymphoma 34:231-239). This region is deleted in a subset ofprostate (approximately 50%), ovarian (approximately 30%) and leukemias(approximately 30%). Therefore, Bcl-G can function as a tumorsuppressor. Furthermore, the presence or absence of Bcl-G nucleic acidor polypeptide or changes in Bcl-G nucleic acid or polypeptideexpression, can serve as a marker for predisposition or progression ofcancer, for example, prostate, ovarian and leukemia. Thus, the inventionBcl-G nucleic acids and/or polypeptides can be used for screening forcancer and/or for developing drug candidates for the treatment ofcancer. Invention Bcl-G nucleic acids and/or polypeptides can also beused for discovery of drugs, as disclosed herein, that suppressautoimmunity, inflammation, allergy, allograph rejection, sepsis, andother diseases, including inflammatory diseases.

A new member of the Bcl-2 family was identified, Bcl-G (see Examples).The human BCL-G gene consists of 6 exons, resides on chromosome 12p12,and encodes two proteins through alternative mRNA splicing: Bcl-G (long)and Bcl-G (short) consisting of 327 and 252 (length) amino acids,respectively. Bcl-G_(L) and Bcl-G_(S) are identical in their first 226amino-acids but diverge thereafter. Among the Bcl-2 Homology (BH)domains previously recognized in Bcl-2 family proteins, the BH3 domainis found in both Bcl-G_(L) and Bcl-G_(S), but only the longer Bcl-G_(L)protein possesses a BH2 domain. Bcl-G_(L) mRNA is expressed widely innormal human tissues, whereas Bcl-G_(S) mRNA was found only in testis.Over-expression of Bcl-G_(L) or Bcl-G_(S) in cells induced apoptosis,but Bcl-G_(S) was far more potent than Bcl-G_(L). Apoptosis induction byBcl-GS depended on the BH3 domain, and was suppressed by co-expressionof anti-apoptotic Bcl-XL protein. Bcl-XL also co-immunoprecipitated withBcl-G_(S) but not with mutants of Bcl-G_(S) in which the BH3 domain wasdeleted or mutated and not with Bcl-G_(L). Bcl-G_(S) was predominantlylocalized to cytosolic organelles whereas Bcl-G_(L) was diffuselydistributed throughout the cytosol. The findings suggest that Bcl-G_(L)is likely in a latent state, whereas the shorter Bcl-G_(S) protein isconstitutively active.

The term “biologically active” or “functional”, when used herein as amodifier of an invention Bcl-G, or polypeptide fragment thereof, refersto a polypeptide that exhibits functional characteristics similar toBcl-G, including those disclosed herein (see Examples I-IX). Asdisclosed herein, Bcl-G induces apoptosis (see Example IV). Therefore,one function of Bcl-G is a pro-apoptotic function. The pro-apoptoticfunction of Bcl-G is inhibited by co-expression of the anti-apoptoticprotein Bcl-2 (Example IV). Therefore, another function of Bcl-G ismodulation by or interaction with an anti-apoptotic protein such as forexample, Bcl-2 family member, including Bcl-2 or Bcl-X_(L), and thelike. Bcl-G can function to heterodimerize with a Bcl-2 family member,thereby modulating the apoptotic activity of Bcl-G and/or the Bcl-2family member. For example, the interaction of BCl-G_(S) with Bcl-X_(L)was found to be BH3 domain dependent, and, thus, the pro-apoptoticactivity of Bcl-G_(S) correlates with its ability to bind Bcl-X_(L) (seeExample VI).

Bcl-G is also contemplated herein as having the ability to function asan ion channel. Additionally, Bcl-G is contemplated herein as having theability to function target to mitochondria, for example, for example, bybinding directly to mitochondria or via binding to a protein that isassociated with mitochondria such as Bcl-2 or Bcl-X_(L). Bcl-G can alsofunction to bind adenine nucleotide transporter (ANT) and to otherproteins such as voltage-dependent anion channel (VDAC).

Because Bcl-G is located on chromosome 12 in a region that is frequentlydeleted in cancer cells (Example II) it is contemplated herein thatBcl-G functions as a tumor suppressor. Another functional activity ofBcl-G is the ability to act as an immunogen for the production ofpolyclonal and monoclonal antibodies that bind specifically to aninvention Bcl-G. Thus, an invention nucleic acid encoding Bcl-G willencode a polypeptide specifically recognized by an antibody that alsospecifically recognizes the Bcl-G protein including the amino acidsequence, set forth in SEQ ID NOS:2, 4 or 42. Such immunologic activitycan be assayed by any method known to those of skill in the art.Therefore, Bcl-G functional fragments include polypeptide fragments thatfunction as immunogens for generating a Bcl-G-specific antibody andfragments that specifically bind to a Bcl-G-specific antibody.

Bcl-2 family proteins are central regulators of apoptosis (reviewed inReed, J. C., Nature, 387:773-776 (1997); Adams & Cory, Science,281:1322-1326 (1998); Gross et al., Genes Dev., 13:1899-1911 (1999)).Bcl-2 family proteins are conserved throughout the animal kingdom, withhomologues identified in both vertebrates and invertebrates. Theseproteins contain up to four conserved Bcl-2 Homology (BH) domains, BH1,BH2, BH3, and BH4, which are recognized by their amino-acid sequencesimilarity. Both anti-apoptotic and pro-apoptotic Bcl-2 family proteinshave been identified. These proteins control cell life-death decisionsthrough their effects on events such as mitochondrial release ofproteins involved in activation of caspase-family cell death proteases(reviewed in Gross et al., Genes Dev., 13:1899-1911 (1999); Green &Reed, Science, 281:1309-1312 (1998); Kroemer & Reed, Nature Medicine,6:513-519 (2000)). Many Bcl-2 family proteins are capable of physicallyinteracting with each other, forming a complex network of homo- andheterodimers, and these physical interactions sometimes play importantroles in the opposing effects of pro- and anti-apoptotic members of thefamily.

The pro-apoptotic members of the Bcl-2 family can be broadly classifiedinto two groups. One group, including Bax, Bak, and Bok in humans,shares structural similarity with the pore-forming domains of certainbacterial toxins and is capable of forming pores in synthetic membranesin vitro (Schendel et al., Cell Death Differ., 5:372-380 (1998);Antonsson et al., Science, 277:370-372 (1997); Schlesinger et al., Proc.Natl. Acad. Sci. USA, 94:11357-11362 (1997); Shimizu et al., J Biol.Chem., 16:12321-12325 (2000)). These protein exhibit cytotoxic effectsindependently of their ability to bind other Bcl-2 family proteins,including Bcl-2 and other cytoprotective members of the family such asBcl-X_(L), Bcl-W, Bfl-1, and Mcl-1. The second group of pro-apoptoticBcl-2 family proteins varies widely in their amino-acid sequences, oftencontaining only a single region of similarity, the BH3 domain. TheseΔBH3-only@ proteins appear to possess no intrinsic or autonomouscytodestructive activity, and instead operate as trans-dominantinhibitors of the survival proteins. Their antagonism of proteins suchas Bcl-2 and Bcl-X_(L) depends on binding via their BH3 domains to ahydrophobic pocket on target anti-apoptotic proteins (Kelekar &Thompson, Trends Cell Biol., 8:324-330 (1998)).

Gene knock-out studies in mice have demonstrated non-redundant roles forvarious Bcl-2 family genes in regulating cell life and death in specifictissues or under particular physiological or pathological circumstances(Veis et al., Cell 75:229-240 (1993); Motoyama et al., Science,267:1506-1510 (1995); Knudson et al., Science, 270:96-99 (1995);Bouillet et al., Science, 286:1735-8 (1999); Yin et al., Nature,400:886-891 (1999)). Thus, it is important to identify all members ofthe Bcl-2 family and to delineate the cellular contexts in which theycontribute to apoptosis regulation. As disclosed herein, a new member ofthe Bcl-2 family, Bcl-G, has been cloned and characterized.

The nucleic acid molecules described herein are useful for producinginvention proteins, when such nucleic acids are incorporated into avariety of protein expression systems known to those of skill in theart. In addition, such nucleic acid molecules or fragments thereof canbe labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aninvention Bcl-G gene or mRNA transcript in a given sample. The nucleicacid molecules described herein, and fragments thereof, are also usefulas primers and/or templates in a PCR reaction for amplifying genesencoding invention proteins described herein.

The term “nucleic acid”, also referred to as polynucleotides,encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers and can be single stranded ordouble stranded. DNA can be either complementary DNA (cDNA) or genomicDNA, and can represent the sense strand, the anti-sense strand or both.Examples of nucleic acids are RNA, cDNA, or isolated genomic DNAencoding an Bcl-G polypeptide. Such nucleic acids include, but are notlimited to, nucleic acids comprising substantially the same nucleotidesequence as set forth in SEQ ID NOS:1, 3 or 41. In general, a genomicsequence of the invention includes regulatory regions such as promoters,enhancers, and introns that are outside of the exons encoding a Bcl-Gbut does not include proximal genes that do not encode Bcl-G.

Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment.

As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in any of SEQ ID NOS:2, 4 or 42. In anotherembodiment, DNA having “substantially the same nucleotide sequence” asthe reference nucleotide sequence has at least 60% identity with respectto the reference nucleotide sequence. DNA having substantially the samenucleotide sequence can have at least 70%, at least 90%, or at least 95%identity to the reference nucleotide sequence.

As used herein, a “modification” of a nucleic acid can also include oneor several nucleotide additions, deletions, or substitutions withrespect to a reference sequence. A modification of a nucleic acid caninclude substitutions that do not change the encoded amino acid sequencedue to the degeneracy of the genetic code. Such modifications cancorrespond to variations that are made deliberately, or which occur asmutations during nucleic acid replication.

Exemplary modifications of the recited Bcl-G sequences include sequencesthat correspond to homologs of other species, including mammalianspecies such as mouse, primates, including monkey and baboon, rat,rabbit, bovine, porcine, ovine, canine, feline, or other animal species.The corresponding Bcl-G sequences of non-human species can be determinedby methods known in the art, such as by PCR or by screening genomic,cDNA or expression libraries.

Another exemplary modification of the invention Bcl-G can correspond tosplice variant forms of the Bcl-G nucleotide sequence. Additionally, amodification of a nucleotide sequence can include one or more non-nativenucleotides, having, for example, modifications to the base, the sugar,or the phosphate portion, or having a modified phosphodiester linkage.Such modifications can be advantageous in increasing the stability ofthe nucleic acid molecule.

Furthermore, a modification of a nucleotide sequence can include, forexample, a detectable moiety, such as a radiolabel, a fluorochrome, aferromagnetic substance, a luminescent tag or a detectable binding agentsuch as biotin. Such modifications can be advantageous in applicationswhere detection of a Bcl-G nucleic acid molecule is desired.

The invention also encompasses nucleic acids which differ from thenucleic acids shown in SEQ ID NOS: 1, 3 or 41, but which have the samephenotype. Phenotypically similar nucleic acids are also referred to as“functionally equivalent nucleic acids”. As used herein, the phrase“functionally equivalent nucleic acids” encompasses nucleic acidscharacterized by slight and non-consequential sequence variations thatwill function in substantially the same manner to produce the sameprotein product(s) as the nucleic acids disclosed herein. In particular,functionally equivalent nucleic acids encode polypeptides that are thesame as those encoded by the nucleic acids disclosed herein or that haveconservative amino acid variations. For example, conservative variationsinclude substitution of a non-polar residue with another non-polarresidue, or substitution of a charged residue with a similarly chargedresidue. These variations include those recognized by skilled artisansas those that do not substantially alter the tertiary structure of theprotein.

Further provided are nucleic acids encoding Bcl-G polypeptides that, byvirtue of the degeneracy of the genetic code, do not necessarilyhybridize to the invention nucleic acids under specified hybridizationconditions. As used herein, the term “degenerate” refers to codons thatdiffer in at least one nucleotide from a reference nucleic acid, butencode the same amino acids as the reference nucleic acid. Nucleic acidsencoding the invention Bcl-G polypeptides can be comprised ofnucleotides that encode substantially the same amino acid sequence asset forth in SEQ ID NOS:2, 4 or 42.

The invention provides an isolated nucleic acid encoding a Bcl-Gpolypeptide, or a functional fragment thereof. The invention alsoprovides an isolated nucleic acid encoding a Bcl-G polypeptide, or afunctional fragment thereof, comprising a nucleic acid selected from:

-   -   (a) nucleic acid encoding the amino acid sequence set forth in        SEQ ID NOS:2, 4 or 42, or    -   (b) nucleic acid that hybridizes to the nucleic acid of (a)        under moderately stringent conditions, wherein said nucleic acid        contiguously encodes biologically active Bcl-G, or    -   (c) nucleic acid degenerate with respect to either (a) or (b)        above, wherein said nucleic acid encodes biologically active        Bcl-G.

In one embodiment, preferred Bcl-G polypeptide include a long formtermed Bcl-G_(L) and a short form termed Bcl-G_(S). Bcl-G_(L) contains aBH3 and a BH2 domain, whereas Bcl-G_(S) contains only a BH3 domain.Bcl-G_(S) has been found to possess pro-apoptotic activity similar toBax (see Example III).

Hybridization refers to the binding of complementary strands of nucleicacid, for example, sense:antisense strands or probe:target-nucleic acidto each other through hydrogen bonds, similar to the bonds thatnaturally occur in chromosomal DNA. Stringency levels used to hybridizea given probe with target-DNA can be readily varied by those of skill inthe art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-nucleic acid to bind a complementarynucleic acid. The hybridized nucleic acids will generally have at leastabout 60% identity, at least about 75% identity, more at least about 85%identity; or at least about 90% identity. Moderately stringentconditions are conditions equivalent to hybridization in 50% formamide,5×Denhart's solution, 5×SSPE, 0.2% SDS at 42EC, followed by washing in0.2×SSPE, 0.2% SDS, at 42EC.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65EC, for example, if a hybrid is notstable in 0.018M NaCl at 65EC, it will not be stable under highstringency conditions, as contemplated herein. High stringencyconditions can be provided, for example, by hybridization in 50%formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS at 42EC, followed bywashing in 0.1×SSPE, and 0.1% SDS at 65EC.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5×Denhart's solution,6×SSPE, 0.2% SDS at 22EC, followed by washing in 1×SSPE, 0.2% SDS, at37EC. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodiumphosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodiumchloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitablemoderate stringency and high stringency hybridization buffers andconditions are well known to those of skill in the art and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y.(1989); and Ausubel et al., supra, 1999). Nucleic acids encodingpolypeptides hybridize under moderately stringent or high stringencyconditions to substantially the entire sequence, or substantialportions, for example, typically at least 15-30 nucleotides of thenucleic acid sequence set forth in SEQ ID NOS:1, 3 or 41.

The invention also provides a modification of a Bcl-G nucleotidesequence that hybridizes to a Bcl-G nucleic acid molecule, for example,a nucleic acid molecule referenced as SEQ ID NOS:1, 3 or 41, undermoderately stringent conditions. Modifications of Bcl-G nucleotidesequences, where the modification has at least 60% identity to a Bcl-Gnucleotide sequence, are also provided. The invention also providesmodification of a Bcl-G nucleotide sequence having at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity, or at least 95%identity.

Identity of any two nucleic acid sequences can be determined by thoseskilled in the art based, for example, on a BLAST 2.0 computeralignment, using default parameters. BLAST 2.0 searching is available athttp://www.ncbi.nlm.nih.gov/gorf/b12.html., as described by Tatiana etal., FEMS Microbiol Lett. 174:247-250 (1999); Altschul et al., NucleicAcids Res., 25:3389-3402 (1997).

One means of isolating a nucleic acid encoding a Bcl-G polypeptide is toprobe a cDNA library or genomic library with a natural or artificiallydesigned nucleic acid probe using methods well known in the art. Nucleicacid probes derived from the Bcl-G gene are particularly useful for thispurpose. DNA and cDNA molecules that encode Bcl-G polypeptides can beused to obtain complementary genomic DNA, cDNA or RNA from mammals, forexample, human, mouse, rat, rabbit, pig, and the like, or other animalsources, or to isolate related cDNA or genomic clones by the screeningof cDNA or genomic libraries, by methods well known in the art (see, forexample, Sambrook et al., supra, 1989; Ausubel et al., supra, 1999).

The invention additionally provides a nucleic acid that hybridizes underhigh stringency conditions to the Bcl-G coding portion of any of SEQ IDNOS:1, 3 or 41. The invention also provides a nucleic acid having anucleotide sequence the same or substantially the same as set that forthin any of SEQ ID NOS:1, 3 or 41.

The invention also provides a method for identifying nucleic acidsencoding a mammalian Bcl-G by contacting a sample containing nucleicacids with one or more Bcl-G oligonucleotides, wherein the contacting iseffected under high stringency hybridization conditions, and identifyinga nucleic acid that hybridizes to the oligonucleotide. The inventionadditionally provides a method of detecting a Bcl-G nucleic acidmolecule in a sample by contacting the sample with two or more Bcl-Goligonucleotides, amplifying a nucleic acid molecule, and detecting theamplification. The amplification can be performed, for example, usingPCR. The invention further provides oligonucleotides that function assingle stranded nucleic acid primers for amplification of a Bcl-Gnucleic acid, wherein the primers comprise a nucleic acid sequencederived from the nucleic acid sequences set forth as SEQ ID NOS:1, 3 or41.

In accordance with a further embodiment of the present invention,optionally labeled Bcl-G-encoding nucleic acids, or fragments thereof,can be employed to probe a library, for example, a cDNA or genomiclibrary, and the like for additional nucleic acid sequences encodingnovel Bcl-G polypeptides. Construction of suitable cDNA libraries iswell-known in the art. Screening of such a cDNA library is initiallycarried out under low-stringency conditions, which comprise atemperature of less than about 42EC, a formamide concentration of lessthan about 50%, and a moderate to low salt concentration.

Presently preferred probe-based screening conditions comprise atemperature of about 37EC, a formamide concentration of about 20%, and asalt concentration of about 5× sodium chloride, sodium citrate (SSC;20×SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Suchconditions will allow the identification of sequences having asubstantial degree of similarity with the probe sequence, withoutrequiring perfect identity. The phrase “substantial similarity” refersto sequences which share at least 50% identity. Hybridization conditionsare selected which allow the identification of sequences having at least70% identity with the probe, while discriminating against sequenceshaving a lower degree of identity with the probe. As a result, nucleicacids having substantially the same nucleotide sequence as SEQ ID NOS:1,3 or 41 are obtained.

As used herein, a nucleic acid “probe” is single-stranded nucleic acid,or analogs thereof, that has a sequence of nucleotides that includes atleast 14, at least 20, at least 50, at least 100, at least 200, at least300, at least 400, or at least 500 contiguous bases that are the same asor the complement thereof, any contiguous bases set forth in any of SEQID NOS:1, 3 or 41. In addition, the entire cDNA encoding region of aninvention Bcl-G, or the entire sequence corresponding to SEQ ID NOS:1, 3or 41 can be used as a probe. Probes can be labeled by methodswell-known in the art, as described hereinafter, and used, for example,in various diagnostic kits.

The invention additionally provides a Bcl-G oligonucleotide comprisingbetween 15 and 300 contiguous nucleotides of SEQ ID NOS:1, 3 or 41, orthe anti-sense strand thereof. As used herein, the term“oligonucleotide” refers to a nucleic acid molecule that includes atleast 15 contiguous nucleotides from a reference nucleotide sequence,can include at least 16, 17, 18, 19, or at least 25 contiguousnucleotides, and often includes at least 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, up to 350 contiguousnucleotides from the reference nucleotide sequence. The referencenucleotide sequence can be the sense strand or the anti-sense strand.

The Bcl-G oligonucleotides of the invention that contain at least 15contiguous nucleotides of a reference Bcl-G nucleotide sequence are ableto hybridize to Bcl-G under moderately stringent hybridizationconditions and thus can be advantageously used, for example, as probesto detect Bcl-G DNA or RNA in a sample, and to detect splice variantsthereof; as sequencing or PCR primers; as antisense reagents to blocktranscription of Bcl-G RNA in cells; or in other applications known tothose skilled in the art in which hybridization to a Bcl-G nucleic acidmolecule is desirable.

It is understood that a Bcl-G nucleic acid molecule, as used herein,specifically excludes previously known nucleic acid molecules consistingof nucleotide sequences having identity with the Bcl-G nucleotidesequence (SEQ ID NOS:1, 3 or 41), such as Expressed Sequence Tags(ESTs), Sequence Tagged Sites (STSs) and genomic fragments, deposited inpublic databases such as the nr, dbest, dbsts, gss and htgs databases,which are available for searching athttp://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=0, using the programBLASTN 2.0.9 described by Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).

In particular, a Bcl-G nucleic acid molecule specifically excludesnucleic acid molecules consisting of any of the nucleotide sequenceshaving the Genbank (gb), EMBL (emb) or DDBJ (dbj) accession numbersdescribed below. Similarly, a Bcl-G polypeptide fragment specificallyexcludes the amino acid fragments encoded by the nucleotide sequenceshaving the GenBank accession numbers described below. GenBank accessionnumbers specifically excluded include AC005903, AC007439, AW000827,AA399486, AW001213, AI478889, AA400686, AA398276, AI240211, andAA536718. The human BAC referenced as GenBank accession No. AC007537 isalso specifically excluded from a Bcl-G nucleic acid.

The isolated Bcl-G nucleic acid molecules of the invention can be usedin a variety of diagnostic and therapeutic applications. For example,the isolated Bcl-G nucleic acid molecules of the invention can be usedas probes, as described above; as templates for the recombinantexpression of Bcl-G polypeptides; or in screening assays such astwo-hybrid assays to identify cellular molecules that bind Bcl-G.

Another useful method for producing a Bcl-G nucleic acid molecule of theinvention involves amplification of the nucleic acid molecule using PCRand Bcl-G oligonucleotides and, optionally, purification of theresulting product by gel electrophoresis. Either PCR or RT-PCR can beused to produce a Bcl-G nucleic acid molecule having any desirednucleotide boundaries. Desired modifications to the nucleic acidsequence can also be introduced by choosing an appropriateoligonucleotide primer with one or more additions, deletions orsubstitutions. Such nucleic acid molecules can be amplifiedexponentially starting from as little as a single gene or mRNA copy,from any cell, tissue or species of interest.

The invention thus provides methods for detecting Bcl-G nucleic acid ina sample. The methods of detecting Bcl-G nucleic acid in a sample can beeither qualitative or quantitative, as desired. For example, thepresence, abundance, integrity or structure of a Bcl-G can bedetermined, as desired, depending on the assay format and the probe usedfor hybridization or primer pair chosen for application.

Useful assays for detecting Bcl-G nucleic acid based on specifichybridization with an isolated Bcl-G nucleic acid molecule are wellknown in the art and include, for example, in situ hybridization, whichcan be used to detect altered chromosomal location of the nucleic acidmolecule, altered gene copy number, and RNA abundance, depending on theassay format used. Other hybridization assays include, for example,Northern blots and RNase protection assays, which can be used todetermine the abundance and integrity of different RNA splice variants,and Southern blots, which can be used to determine the copy number andintegrity of DNA. A Bcl-G hybridization probe can be labeled with anysuitable detectable moiety, such as a radioisotope, fluorochrome,chemiluminescent marker, biotin, or other detectable moiety known in theart that is detectable by analytical methods.

Useful assays for detecting a Bcl-G nucleic acid in a sample based onamplifying a Bcl-G nucleic acid with two or more Bcl-G oligonucleotidesare also well known in the art, and include, for example, qualitative orquantitative polymerase chain reaction (PCR); reverse-transcriptionPCR(RT-PCR); single strand conformational polymorphism (SSCP) analysis,which can readily identify a single point mutation in DNA based ondifferences in the secondary structure of single-strand DNA that producean altered electrophoretic mobility upon non-denaturing gelelectrophoresis; and coupled PCR, transcription and translation assays,such as a protein truncation test, in which a mutation in DNA isdetermined by an altered protein product on an electrophoresis gel.Additionally, the amplified Bcl-G nucleic acid can be sequenced todetect mutations and mutational hot-spots, and specific assays forlarge-scale screening of samples to identify such mutations can bedeveloped.

The invention further provides an isolated Bcl-G polypeptide, or afunctional fragment thereof, encoded by a Bcl-G nucleic acid of theinvention. For example, the invention provides a polypeptide comprisingthe same or substantially the same amino acid sequence as Bcl-G_(L) (SEQID NO:2) or Bcl-G_(S) (SEQ ID NO:4). Also provided is a Bcl-Gpolypeptide encoded by a nucleotide sequence comprising the same orsubstantially the same nucleotide sequence as set forth in SEQ ID NOs:1or 3. Additionally provided is mouse Bcl-G nucleotide sequence set forthas SEQ ID NO:41 (see Example IX).

Described herein is a new member of the BCL-2 gene family in humans,BCL-G (see Examples I-VIII). The BCL-G gene potentially encodes twoprotein products, Bcl-G_(L) and Bcl-G_(S). Bcl-2 family proteins containup to four conserved BH domains. The shorter Bcl-G_(S) protein containsonly the BH3 domains, similar to several other pro-apoptotic Bcl-2family proteins, including Bad, Hrk, Bik, Bim, Apr, and Egll (reviewedin Kelekar & Thompson, Trends Cell Biol., 8:324-330 (1998); Reed, J.Oncogene, 17:3225-3236 (1998)). In contrast, the longer Bcl-G_(L)protein contains a BH2 and BH3 domain. No other examples of Bcl-2 familyproteins are known which combine BH2 and BH3 domain in the absence ofBH1. Though the Bad protein was originally suggested to contain a BH2domain (Yang et al., Blood, 84(Suppl.1):373a-380a (1994)), and has beenshown to possess the BH3 domain, inspection of the BH2 region revealsvery little similarity of amino-acid sequence with (Ottilie et al., J.Biol. Chem., 272:30866-30872 (1997)) other BH2 domains. In contrast, theBH2 of Bcl-G_(L) contains a stretch of 8 of 8 residues showing identityor conservative amino-acid substitutions with the BH2 domains of otherfamily members. By comparison, the Bad sequence reveals only 3 of 8identical or similar amino-acids in the same region. Thus, Bcl-G_(L)defines a novel structural variant within the Bcl-2 family ofapoptosis-regulating proteins.

The production of different protein isoforms by alternative mRNAsplicing is a common feature of BCL-2 family genes, including BCL-2,Bcl-X, MCL-1, BAX, and BIM (Tsujimoto & Croce, Proc. Natl. Acad. Sci.USA, 83:5214-5218 (1986); O'Connor et al., EMBO 117:384-395 (1998);Boise et al., Cell, 74:597-608 (1993); Oltvai et al., Cell, 74:609-619(1993); Bingle et al., J. Biol. Chem., 275:22136-22146 (2000)). UnlikeBCL-X, which encodes a longer and short protein, Bcl-X_(L) andBcl-X_(S), possessing anti-apoptotic and pro-apoptotic functions,respectively, the longer isoform of Bcl-G did not display anti-apoptoticactivity. When over-expressed, Bcl-G_(L) induced modest and variableincreases in apoptosis, whereas the shorter Bcl-G_(S) proteinconsistently exhibited potent cytotoxic activity. This behavior isreminiscent of the proteins encoded by the BIM gene, which includeBim-short (Bim_(S)), Bim-long (Bim_(L)) and Bim-Extra-Long (Bim_(EL))(O'Connor et al., EMBO J., 17:384-395 (1998)). The longer proteins,Bim_(L) and Bim_(EL), are sequestered in complexes with dyneinlight-chain (DLC) in association with microtubules, thus preventing themfrom interacting with target proteins such as Bcl-X_(L) on the surfaceof mitochondria and other organelles (Puthalakath et al., Mol. Cell,3:287-96 (1999)). In contrast, because the shortest isoform, Bim_(S),does not associate with DLC, it is free to interact with Bcl-X_(L),Bcl-2, and other survival proteins and hence displays far more potentapoptotic activity when over-expressed in cells. By analogy, the longerBcl-G_(L) protein could be sequestered in an inactive complex with anunidentified protein.

Besides interactions with sequestering proteins, the activity ofpro-apoptotic Bcl-2 family proteins can be suppressed by othermechanisms, including post-translational modifications. For example, theBad protein is inactivated by phosphorylation. This protein can bedirectly or indirectly phosphorylated by several protein kinases,including PKA, PKB (Akt), Raf1, and Pak1, thus preventing it fromdimerizing with target proteins such as Bcl-2 and Bcl-X_(L) (reviewed inReed, J. Oncogene, 17:3225-3236 (1998); Datta et al., Genes Dev.,13:2905-2927 (1999)). The intracellular location of Bad varies,depending on its phosphorylation state, with phosphorylated Bad residingin the cytosol and unphosphorylated Bad associated with mitochondria andother intracellular organelles where Bcl-2 and Bcl-X_(L) are located. Inthis regard, the Bcl-G_(L) protein contains candidate phosphorylationsites for protein kinase A (PKA) and protein kinase C (PKC), includingsome not found in Bcl-G_(S). However, in vivo phosphorylation ofBcl-G_(L) has not been observed in pilot experiments.

Another post-translational modification shown previously to activatelatent pro-apoptotic Bcl-2 family proteins is proteolysis. Specifically,the Bid protein contains a N-terminal domain of ˜56 amino-acids thatmasks its BH3 domain, reducing its ability to dimerize with other Bcl-2family proteins. Upon cleavage by caspases, however, removal of theN-terminal domain exposes the BH3 domain and is associated withtranslocation of Bid from the cytosol to mitochondria, where it inducescytochrome c release and apoptosis (Li et al., Cell, 94:491-501 (1998);Luo et al., Cell, 94:481-490 (1998)). While Bcl-G_(L) contains candidatecaspase recognition sites, no significant cleavage of Bid has beenobserved in vitro using purified active caspases or in cells duringapoptosis. It is possible, however, that a specific caspase not yettested is capable of cleaving and activating Bcl-G_(L).

Though possessing no hydrophobic region that might anchor it inmembranes, the Bcl-G_(S) protein was constitutively associated withintracellular organelles. Interestingly, removal of the BH3 domain didnot interfere with organellar-targeting of Bcl-G_(S), but did abolishdimerization with Bcl-X_(L). Thus, the BH3 domain apparently is notresponsible for association of Bcl-G_(S) with intracellular organelles.This BH3-independent targeting of Bcl-G_(S) differs from some other“BH3-only” Bcl-2 family proteins such as Bad, where it has been observedthat removal of the BH3 domain abrogates binding to anti-apoptotic Bcl-2family proteins as well as association with mitochondria (Zha et al., J.Biol. Chem., 272:24101-24104 (1997)).

The BCL-G gene resides on chromosome 12p12, a region deleted in ˜50% ofprostate cancers, ˜30% of ovarian cancers, and ˜30% of childhood acutelymphocytic leukemias (ALLs) (Kibel et al., J. Urol., 1:192-196 (2000);Aissani et al., Leuk Lymphoma, 34:231-239 (1999); Hatta et al., Br JCancer, 75:1256-1262 (1997)). Given that at least one of the proteinproducts of the BCL-G gene exhibits pro-apoptotic function, it ispossible that BCL-G represents a tumor suppressor gene. However, thusfar, somatic mutations in the exons of BCL-G have not been detected norevidence of deletion of both BCL-G alleles in tumor cell lines orprimary tumor specimens tested so far. Further studies are requiredtherefore to determine whether loss of BCL-G expression occurs in tumorsby means other than somatic alterations in gene structure and DNAsequence, such as changes in gene methylation or aberranttranscriptional or post-transcriptional regulation.

Investigation of the tissue-distribution of Bcl-G_(L) and Bcl-G_(S)mRNAs by RT-PCR revealed that Bcl-G_(L) mRNA is found in several normaladult tissues, whereas Bcl-G_(S) was detected only in testis. Thisfinding indicates tissue-specific regulation of Bcl-G_(S) mRNA splicing.Tissue-specific splicing of other Bcl-2 family mRNAs has been observedpreviously. For example, Bcl-X mRNA splicing events which generate thepro-apoptotic Bcl-X_(S) protein occur in the thymus during T-cellontogeny and in the mammary gland during post-lactation involution, inassociation with extensive apoptosis induction (Boise et al., Cell,74:597-608 (1993); Heermeier et al., Mech. Dev., 56:197-207 (1996)).Additional studies are performed to assess differential mRNA splicingpatterns of Bcl-G transcripts during fetal development and followingvarious scenarios in the adult where apoptosis occurs as part of anormal physiological response or an abnormal pathological reaction toenvironmental insults.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred. It is recognized,however, that polypeptides, or encoding nucleic acids, containing lessthan the described levels of sequence identity arising as splicevariants or that are modified by conservative amino acid substitutions,or by substitution of degenerate codons are also encompassed within thescope of the present invention.

Also encompassed by the term Bcl-G are functional fragments orpolypeptide analogs thereof. The term “functional fragment” refers to apeptide fragment that is a portion of a full length Bcl-G protein,provided that the portion has a biological activity, as defined herein,that is characteristic of the corresponding full length protein. Thus,the invention also provides functional fragments of invention Bcl-Gproteins, which can be identified using the binding and routine methods,such as bioassays described herein. A Bcl-G polypeptide functionalfragment can be a BH3 or BH2 domain, for example, a BH3 domainreferenced as SEQ ID NOS:5 or 9 or a BH2 domain referenced as SEQ IDNOS:6 or 18. The BH3 domain of Bcl-G is 33% identical to the BH3 domainof Bcl-2, 44% identical to the BH3 domain of Bcl-X_(L), and 66%identical to the BH3 domain of Bax.

In addition, a functional fragment of a Bcl-G polypeptide can be Baxhomology region. A region upstream of the BH3 domain shares a highdegree of homology with Bax, including a 12 amino acid residue motifthat is 70% identical between Bcl-G and Bax. Therefore, such a Baxhomology region can function similarly to Bax, for example, as apossible binding domain. The N-terminal 150 amino acids of Bcl-G are notsimilar to any known amino acid sequence available in public databases.Therefore, the N-terminal region of Bcl-G can function as Bcl-G-specificfunctional domain that confers a biological activity that is specificfor Bcl-G relative to other members of the Bcl-2 family.

The invention also provides a chimeric protein comprising a domainselected from the group consisting of BH3 (SEQ ID NOS:5 or 9) and BH2(SEQ ID NOS:6 or 18). A chimeric protein comprising a Bcl-G functionaldomain can be generated, for example, by recombinantly expressing aBcl-G domain such as BH2 or BH3 fused to another polypeptide.Alternatively, the Bcl-G functional domain can be expressed as a fusionto another polypeptide.

In another embodiment of the invention, Bcl-G-containing chimericproteins are provided comprising an invention Bcl-G, or fragmentsthereof, having the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:42, and further comprising one or more sequences from a heterologousprotein. Sequences from heterologous proteins with which the Bcl-G orfunctional fragment thereof are fused can include, for example,glutathione-S-transferase, an antibody, or other proteins or functionalfragments thereof which facilitate recovery of the chimera. Furtherproteins with which the Bcl-G or functional fragment thereof are fusedwill include, for example, luciferase, green fluorescent protein, anantibody, or other proteins or functional fragments thereof whichfacilitate identification of the chimera. Still further proteins withwhich the Bcl-G or functional fragment thereof are fused will include,for example, the LexA DNA binding domain, ricin, a-sarcin, an antibody,or other proteins which have therapeutic properties or other biologicalactivity.

As such chimeric proteins include sequences from two different proteins,the resultant amino acid sequence of the chimeric protein will typicallybe a non-naturally occurring sequence. Thus, in accordance with thisembodiment of the invention, there are provided chimeric proteinscomprising an invention Bcl-G, or fragments thereof, having the sequenceof SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:42, or a fragment thereof,provided the sequence of the chimeric protein is not naturallyoccurring.

In another embodiment of the invention, there are providedhetero-oligomers comprising invention Bcl-G polypeptides and fragmentsthereof, invention Bcl-G-containing proteins, Bcl-G-containing chimericproteins, or combinations thereof. As disclosed herein, Bcl-G contains aBH3 domain, which functions as a ligand to bind Bcl-2 family members(Example I). Bcl-G can function to bind Bcl-2 family members. Thus,hetero-oligomers comprising invention Bcl-G polypeptides (SEQ ID NOS:2,4 or 42) and fragments thereof, invention Bcl-G-containing proteins,Bcl-G-containing chimeric proteins, or combinations thereof, and furthercomprising Bcl-2 family members such as Bcl-2, Bcl-X_(L) or other Bcl-2family members are provided.

As used herein, the term “polypeptide” when used in reference to Bcl-Gis intended to refer to a peptide or polypeptide of two or more aminoacids. The term “polypeptide analog” includes any polypeptide having anamino acid residue sequence substantially the same as a sequencespecifically described herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the ability to functionally mimic a Bcl-G as described herein.A Amodification@ of a Bcl-G polypeptide also encompasses conservativesubstitutions of a Bcl-G polypeptide amino acid sequence. Conservativesubstitutions of encoded amino acids include, for example, amino acidsthat belong within the following groups: (1) non-polar amino acids (Gly,Ala, Val, Leu, and Ile); (2) polar neutral amino acids (Cys, Met, Ser,Thr, Asn, and Gln); (3) polar acidic amino acids (Asp and Glu); (4)polar basic amino acids (Lys, Arg and His); and (5) aromatic amino acids(Phe, Trp, Tyr, and His). Other minor modifications are included withinBcl-G polypeptides so long as the polypeptide retains some or all of itsfunction as described herein.

The amino acid length of functional fragments or polypeptide anlogs ofthe present invention can range from about 5 amino acids up to thefull-length protein sequence of an invention Bcl-G. In certainembodiments, the amino acid lengths include, for example, at least about10 amino acids, at least about 15, at least about 20, at least about 25,at least about 30, at least about 35, at least about 40, at least about45, at least about 50, at least about 75, at least about 100, at leastabout 150, at least about 200, at least about 250 or more amino acids inlength up to the full-length Bcl-G protein sequence. The functionalfragments can be contiguous amino acid sequences of a Bcl-G polypeptide,including contiguous amino acid sequences of SEQ ID NOS:2, 4 or 42.

A modification of a polypeptide can also include derivatives, analoguesand functional mimetics thereof, provided that such polypeptide displaysthe Bcl-G biological activity. For example, derivatives can includechemical modifications of the polypeptide such as alkylation, acylation,carbamylation, iodination, or any modification that derivatizes thepolypeptide. Such derivatized molecules include, for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups can be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups canbe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine can be derivatized to form N-im-benzylhistidine.Also included as derivatives or analogues are those peptides whichcontain one or more naturally occurring amino acid derivatives of thetwenty standard amino acids, for example, 4-hydroxyproline,5-hydroxylysine, 3-methylhistidine, homoserine, ornithine orcarboxyglutamate, and can include amino acids that are not linked bypeptide bonds. Polypeptides of the present invention also include anypolypeptide having one or more additions and/or deletions of residues,relative to the sequence of a polypeptide whose sequence is shownherein, so long as Bcl-G activity is maintained.

A modification of a Bcl-G polypeptide includes functional mimeticsthereof. Mimetics encompass chemicals containing chemical moieties thatmimic the function of the polypeptide. For example, if a polypeptidecontains two charged chemical moieties having functional activity, amimetic places two charged chemical moieties in a spatial orientationand constrained structure so that the charged chemical function ismaintained in three-dimensional space. Thus, a mimetic, which orientsfunctional groups that provide a function of Bcl-G, are included withinthe meaning of a Bcl-G derivative. All of these modifications areincluded within the term “polypeptide” so long as the Bcl-G polypeptideor functional fragment retains its function.

The invention provides an isolated Bcl-G polypeptide, or functionalfragment thereof. The invention Bcl-G polypeptides can be isolated by avariety of methods well-known in the art, for example, recombinantexpression systems described herein, precipitation, gel filtration,ion-exchange, reverse-phase and affinity chromatography, and the like.Other well-known methods are described in Deutscher et al., Guide toProtein Purification: Methods in Enzymology Vol. 182, (Academic Press,(1990)). Alternatively, the isolated polypeptides of the presentinvention can be obtained using well-known recombinant methods (see, forexample, Sambrook et al., supra, 1989; Ausubel et al., supra, 1999). Themethods and conditions for biochemical purification of a polypeptide ofthe invention can be chosen by those skilled in the art, andpurification monitored, for example, by an immunological assay or afunctional assay.

An example of the means for preparing the invention polypeptide(s) is toexpress nucleic acids encoding Bcl-G in a suitable host cell, such as abacterial cell, a yeast cell, an amphibian cell such as an oocyte, or amammalian cell, using methods well known in the art, and recovering theexpressed polypeptide, again using well-known purification methods, sodescribed herein. Invention polypeptides can be isolated directly fromcells that have been transformed with expression vectors as describedherein. Recombinantly expressed polypeptides of the invention can alsobe expressed as fusion proteins with appropriate affinity tags, such asglutathione S transferase (GST) or poly His, and affinity purified. Theinvention polypeptide, biologically functional fragments, and functionalequivalents thereof can also be produced by chemical synthesis. Forexample, synthetic polypeptides can be produced using AppliedBiosystems, Inc. Model 430A or 431A automatic peptide synthesizer(Foster City, Calif.) employing the chemistry provided by themanufacturer.

Bcl-G polypeptides can be administered to an individual to increase anactivity associated with a Bcl-G polypeptide, including induction ofapoptosis or functioning as a tumor suppressor. For example, a Bcl-Gpolypeptide can be administered therapeutically to an individual usingexpression vectors containing nucleic acids encoding Bcl-G polypeptides,as described below. In addition, Bcl-G polypeptides, or a functionalportion thereof, can be directly administered to an individual. Methodsof administering therapeutic polypeptides are well known to thoseskilled in the art, for example, as a pharmaceutical composition.

In a particular embodiment, a Bcl-G polypeptide, or functional fragmentthereof, can be administered to an individual so that the Bcl-Gpolypeptide or functional fragment is targeted to a tumor to induceapoptosis or otherwise function as a tumor suppressor. One method ofdelivering a Bcl-G polypeptide to an intracellular target is to fuse aBcl-G polypeptide or functional fragment to an intracellular-targetingpeptide that can penetrate the cell membrane or otherwise deliver apolypeptide to the intracellular environment such as viainternalization, thereby causing the fused Bcl-G polypeptide to enterthe cell. One example of such an intracellular-targeting peptides is afusion to the transduction domain of HIV TAT, which allows transductionof up to 100% of cells (Schwarze et al., Science 285:1569-1572 (1999);Vocero-Akbani et al., Nature Med. 5:29-33 (1999)).

Another example of such an intracellular-targeting peptide is theAntennapeida homeoprotein internalization domain (Holinger et al., J.Biol. Chem. 274:13298-13304 (1999)). Still anotherintracellular-targeting peptide is a peptide that is specific for a cellsurface receptor, which allows binding and internalization of a fusionpolypeptide via receptor-mediated endocytosis (Ellerby et al., NatureMed. 5:1032-1038 (1999)). Such intracellular-targeting peptides thatmediate specific receptor interactions can be advantageously used totarget a tumor (see Ellerby et al., supra, 1999). Alternatively, a Bcl-Gpolypeptide of the invention can be incorporated, if desired, intoliposomes, microspheres or other polymer matrices (Gregoriadis, LiposomeTechnology, Vols. I to III, 2nd ed., CRC Press, Boca Raton Fla. (1993)).

The invention additionally provides a method for modulating the activityof an oncogenic polypeptide by contacting the oncogenic polypeptide witha substantially pure Bcl-G, or an oncogenic protein-binding fragmentthereof. Bcl-G can function to bind oncogenic proteins such as Bcl-2.Therefore, Bcl-G or functional fragments that bind to an oncogenicprotein such as Bcl-2 can be used to modulate the activity of theoncogenic protein.

The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified Bcl-G mature proteinor functional polypeptide fragments thereof, alone or in combinationwith each other. These polypeptides or proteins can be recombinantlyderived, chemically synthesized or purified from native sources. As usedherein, the term “acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as phosphate buffered saline solution,water and emulsions such as an oil and water emulsion, and various typesof wetting agents.

The invention thus provides a therapeutic composition comprising apharmaceutically acceptable carrier and a compound selected from thegroup consisting of a Bcl-G polypeptide, a functional fragment of Bcl-G,a Bcl-G modulating compound, and an anti-Bcl-G antibody. The inventionadditionally provides a method of treating a pathology characterized byabnormal cell proliferation by administering an effective amount of thecomposition containing a pharmaceutically acceptable carrier and acompound selected from the group consisting of a Bcl-G polypeptide, afunctional fragment of Bcl-G, a Bcl-G modulating compound, and ananti-Bcl-G antibody.

Also provided are antisense-nucleic acids having a sequence capable ofbinding specifically with full-length or any portion of an mRNA thatencodes Bcl-G polypeptides so as to prevent translation of the mRNA. Theantisense-nucleic acid can have a sequence capable of bindingspecifically with any portion of the sequence of the cDNA encoding Bcl-Gpolypeptides. As used herein, the phrase “binding specifically”encompasses the ability of a nucleic acid sequence to recognize acomplementary nucleic acid sequence and to form double-helical segmentstherewith via the formation of hydrogen bonds between the complementarybase pairs. An example of an antisense-nucleic acid is anantisense-nucleic acid comprising chemical analogs of nucleotides.

The present invention provides means to modulate levels of expression ofBcl-G polypeptides by recombinantly expressing Bcl-G anti-sense nucleicacids or employing synthetic anti-sense nucleic acid compositions(hereinafter SANC) that inhibit translation of mRNA encoding thesepolypeptides. Synthetic oligonucleotides, or other antisense-nucleicacid chemical structures designed to recognize and selectively bind tomRNA are constructed to be complementary to full-length or portions ofan Bcl-G coding strand, including nucleotide sequences set forth in SEQID NOS:1, 3 or 41.

The SANC is designed to be stable in the blood stream for administrationto a subject by injection, or in laboratory cell culture conditions. TheSANC is designed to be capable of passing through the cell membrane inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SANC, which render it capable of passingthrough cell membranes, for example, by designing small, hydrophobicSANC chemical structures, or by virtue of specific transport systems inthe cell which recognize and transport the SANC into the cell. Inaddition, the SANC can be designed for administration only to certainselected cell populations by targeting the SANC to be recognized byspecific cellular uptake mechanisms which bind and take up the SANC onlywithin select cell populations. In a particular embodiment the SANC isan antisense oligonucleotide.

For example, the SANC may be designed to bind to a receptor found onlyin a certain cell type, as discussed above. The SANC is also designed torecognize and selectively bind to target mRNA sequence, which cancorrespond to a sequence contained within the sequences shown in SEQ IDNOS:1, 3 or 41. The SANC is designed to inactivate target mRNA sequenceby either binding thereto and inducing degradation of the mRNA by, forexample, RNase I digestion, or inhibiting translation of mRNA targetsequence by interfering with the binding of translation-regulatingfactors or ribosomes, or inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups which either degrade orchemically modify the target mRNA. SANCs have been shown to be capableof such properties when directed against mRNA targets (see Cohen et al.,TIPS, 10:435 (1989) and Weintraub, Sci. American, January (1990), pp.40).

The invention further provides a method of modulating the level ofapoptosis in a cell by introducing an antisense nucleotide sequence intothe cell, wherein the antisense nucleotide sequence specificallyhybridizes to a nucleic acid molecule encoding a Bcl-G, wherein thehybridization reduces or inhibits the expression of the Bcl-G in thecell. The use of anti-sense nucleic acids, including recombinantanti-sense nucleic acids or SANCs, can be advantageously used to inhibitcell death.

Compositions comprising an amount of the antisense-nucleic acid of theinvention, effective to reduce expression of Bcl-G polypeptides byentering a cell and binding specifically to mRNA encoding Bcl-Gpolypeptides so as to prevent translation and an acceptable hydrophobiccarrier capable of passing through a cell membrane are also providedherein. Suitable hydrophobic carriers are described, for example, inU.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and the like. Theacceptable hydrophobic carrier capable of passing through cell membranesmay also comprise a structure which binds to a receptor specific for aselected cell type and is thereby taken up by cells of the selected celltype. For example, the structure can be part of a protein known to bindto a cell-type specific receptor such as a tumor.

Antisense-nucleic acid compositions are useful to inhibit translation ofmRNA encoding invention polypeptides. Synthetic oligonucleotides, orother antisense chemical structures are designed to bind to mRNAencoding Bcl-G polypeptides and inhibit translation of mRNA and areuseful as compositions to inhibit expression of Bcl-G associated genesin a tissue sample or in a subject.

The invention also provides a method for expression of a Bcl-Gpolypeptide by culturing cells containing a Bcl-G nucleic acid underconditions suitable for expression of Bcl-G. Thus, there is provided amethod for the recombinant production of a Bcl-G of the invention byexpressing the nucleic acid sequences encoding Bcl-G in suitable hostcells. Recombinant DNA expression systems that are suitable to produceBcl-G described herein are well-known in the art (see, for example,Ausubel et al., supra, 1999). For example, the above-describednucleotide sequences can be incorporated into vectors for furthermanipulation. As used herein, vector refers to a recombinant DNA or RNAplasmid or virus containing discrete elements that are used to introduceheterologous DNA into cells for either expression or replicationthereof.

The invention also provides vectors containing the Bcl-G nucleic acidsof the invention. Suitable expression vectors are well-known in the artand include vectors capable of expressing nucleic acid operativelylinked to a regulatory sequence or element such as a promoter region orenhancer region that is capable of regulating expression of such nucleicacid. Appropriate expression vectors include those that are replicablein eukaryotic cells and/or prokaryotic cells and those that remainepisomal or those which integrate into the host cell genome.

Promoters or enhancers, depending upon the nature of the regulation, canbe constitutive or regulated. The regulatory sequences or regulatoryelements are operatively linked to a nucleic acid of the invention suchthat the physical and functional relationship between the nucleic acidand the regulatory sequence allows transcription of the nucleic acid.

Suitable vectors for expression in prokaryotic or eukaryotic cells arewell known to those skilled in the art (see, for example, Ausubel etal., supra, 1999). Vectors useful for expression in eukaryotic cells caninclude, for example, regulatory elements including the SV40 earlypromoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumorvirus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus(MMLV) promoter, and the like. The vectors of the invention are usefulfor subcloning and amplifying a Bcl-G nucleic acid molecule and forrecombinantly expressing a Bcl-G polypeptide. A vector of the inventioncan include, for example, viral vectors such as a bacteriophage, abaculovirus or a retrovirus; cosmids or plasmids; and, particularly forcloning large nucleic acid molecules, bacterial artificial chromosomevectors (BACs) and yeast artificial chromosome vectors (YACs). Suchvectors are commercially available, and their uses are well known in theart. One skilled in the art will know or can readily determine anappropriate promoter for expression in a particular host cell.

The invention additionally provides recombinant cells containing Bcl-Gnucleic acids of the invention. The recombinant cells are generated byintroducing into a host cell a vector containing a Bcl-G nucleic acidmolecule. The recombinant cells are transducted, transfected orotherwise genetically modified. Exemplary host cells that can be used toexpress recombinant Bcl-G molecules include mammalian primary cells;established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3, HEK293 and PC12 cells; amphibian cells, such as Xenopus embryos andoocytes; and other vertebrate cells. Exemplary host cells also includeinsect cells such as Drosophila, yeast cells such as Saccharomycescerevisiae, Saccharomyces pombe, or Pichia pastoris, and prokaryoticcells such as Escherichia coli.

In one embodiment, nucleic acids encoding the invention Bcl-Gpolypeptides can be delivered into mammalian cells, either in vivo or invitro using suitable vectors well-known in the art. Suitable vectors fordelivering a Bcl-G polypeptide, or a functional fragment thereof to amammalian cell, include viral vectors such as retroviral vectors,adenovirus, adeno-associated virus, lentivirus, herpesvirus, as well asnon-viral vectors such as plasmid vectors. Such vectors are useful forproviding therapeutic amounts of a Bcl-G polypeptide (see, for example,U.S. Pat. No. 5,399,346, issued Mar. 21, 1995). Delivery of Bcl-Gpolypeptides or nucleic acids therapeutically can be particularly usefulwhen targeted to a tumor cell, thereby inducing apoptosis in tumorcells. In addition, where it is desirable to limit or reduce the in vivoexpression of the invention Bcl-G, the introduction of the antisensestrand of the invention nucleic acid is contemplated.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing invention nucleic acidencoding an Bcl-G protein into mammalian cells are well known in theart. These viral vectors include, for example, Herpes simplex virusvectors (Geller et al., Science, 241:1667-1669 (1988)); vaccinia virusvectors (Piccini et al., Meth. Enzymology, 153:545-563 (1987));cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzmanand S. H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1988, pp. 78-84)); Moloney murine leukemia virus vectors(Danos et al., Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988); Blaeseet al., Science, 270:475-479 (1995); Onodera et al., J. Virol.,72:1769-1774 (1998)); adenovirus vectors (Berkner, Biotechniques,6:616-626 (1988); Cotten et al., Proc. Natl. Acad. Sci. USA,89:6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991);Li et al., Human Gene Therapy, 4:403-409 (1993); Zabner et al., NatureGenetics, 6:75-83 (1994)); adeno-associated virus vectors (Goldman etal., Human Gene Therapy, 10:2261-2268 (1997); Greelish et al., NatureMed., 5:439-443 (1999); Wang et al., Proc. Natl. Acad. Sci. USA,96:3906-3910 (1999); Snyder et al., Nature Med., 5:64-70 (1999); Herzoget al., Nature Med., 5:56-63 (1999)); retrovirus vectors (Donahue etal., Nature Med., 4:181-186 (1998); Shackleford et al., Proc. Natl.Acad. Sci. USA, 85:9655-9659 (1988); U.S. Pat. Nos. 4,405,712, 4,650,764and 5,252,479, and WIPO publications WO 92/07573, WO 90/06997, WO89/05345, WO 92/05266 and WO 92/14829; and lentivirus vectors (Kafri etal., Nature Genetics, 17:314-317 (1997)).

For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes(Wagner et al., Proc. Natl. Acad. Sci., USA, 89:6099-6103 (1992); Curielet al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum. Gene Ther.,4:14-24 (1993)) are employed to transduce mammalian cells withheterologous Bcl-G nucleic acid. Any of the plasmid expression vectorsdescribed herein may be employed in a TfAdpl-DNA complex.

Vectors useful for therapeutic administration of a Bcl-G polypeptide ofnucleic acid can contain a regulatory element that provides tissuespecific or inducible expression of an operatively linked nucleic acid.One skilled in the art can readily determine an appropriatetissue-specific promotor or enhancer that allows exparssion of a Bcl-Gpolypeptide or nucleic acid in a desired tissue. Any of a variety ofinducible promoters or enhancers can also be included in the vector forregulatable expression of a Bcl-G polypeptide or nucleic acid. Suchinducible systems, include, for example, tetracycline inducible system(Gossen & Bizard, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992);Gossen et al., Science, 268:1766-1769 (1995); Clontech, Palo Alto,Calif.); metalothionein promoter induced by heavy metals; insect steroidhormone responsive to ecdysone or related steroids such as muristerone(No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996); Yao et al.,Nature, 366:476-479 (1993); Invitrogen, Carlsbad, Calif.); mouse mammorytumor virus (MMTV) induced by steroids such as glucocortocoid andestrogen (Lee et al., Nature, 294:228-232 (1981); and heat shockpromoters inducible by temperature changes.

An inducible system particularly useful for therapeutic administrationutilizes an inducible promotor that can be regulated to deliver a levelof therapeutic product in response to a given level of drug administeredto an individual and to have little or no expression of the therapeuticproduct in the absence of the drug. One such system utilizes a Ga14fusion that is inducible by an antiprogestin such as mifepristone in amodified adenovirus vector (Burien et al., Proc. Natl. Acad. Sci. USA,96:355-360 (1999). Another such inducible system utilizes the drugrapamycin to induce reconstitution of a transcriptional activatorcontaining rapamycin binding domains of FKBP12 and FRAP in anadeno-associated virus vector (Ye et al., Science, 283:88-91 (1999)). Itis understood that any combination of an inducible system can becombined in any suitable vector, including those disclosed herein. Sucha regulatable inducible system is advantageous because the level ofexpression of the therapeutic product can be controlled by the amount ofdrug administered to the individual or, if desired, expression of thetherapeutic product can be terminated by stopping administration of thedrug.

The invention additionally provides an isolated anti-Bcl-G antibodyhaving specific reactivity with a Bcl-G. The anti-Bcl-G antibody can bea monoclonal antibody or a polyclonal antibody. The invention furtherprovides cell lines producing monoclongal antibodies having specificreactivity with a Bcl-G.

The invention thus provides antibodies that specifically bind a Bcl-Gpolypeptide. As used herein, the term “antibody” is used in its broadestsense to include polyclonal and monoclonal antibodies, as well asantigen binding fragments of such antibodies. With regard to ananti-Bcl-G antibody of the invention, the term “antigen” means a nativeor synthesized Bcl-G polypeptide or fragment thereof. An anti-Bcl-Gantibody, or antigen binding fragment of such an antibody, ischaracterized by having specific binding activity for a Bcl-Gpolypeptide or a peptide portion thereof of at least about 1×10⁵ M⁻¹.Thus, Fab, F(ab′)₂, Fd and Fv fragments of an anti-Bcl-G antibody, whichretain specific binding activity for a Bcl-G polypeptide, are includedwithin the definition of an antibody. Specific binding activity of aBcl-G polypeptide can be readily determined by one skilled in the art,for example, by comparing the binding activity of an anti-Bcl-G antibodyto a Bcl-G polypeptide versus a control polypeptide that is not a Bcl-Gpolypeptide. Methods of preparing polyclonal or monoclonal antibodiesare well known to those skilled in the art (see, for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1988)).

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al. (Science 246:1275-1281 (1989)). These and other methods ofmaking, for example, chimeric, humanized, CDR-grafted, single chain, andbifunctional antibodies are well known to those skilled in the art(Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al.,Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard etal., Protein Engineering: A practical approach (IRL Press 1992);Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995)).

Anti-Bcl-G antibodies can be raised using a Bcl-G immunogen such as anisolated Bcl-G polypeptide having the amino acid sequence of SEQ IDNOS:2, 4 or 42, or a fragment thereof, which can be prepared fromnatural sources or produced recombinantly, or a peptide portion of theBcl-G polypeptide. Such peptide portions of a Bcl-G polypeptide arefunctional antigenic fragments if the antigenic peptides can be used togenerate a Bcl-G-specific antibody. A non-immunogenic or weaklyimmunogenic Bcl-G polypeptide or portion thereof can be made immunogenicby coupling the hapten to a carrier molecule such as bovine serumalbumin (BSA) or keyhole limpet hemocyanin (KLH). Various other carriermolecules and methods for coupling a hapten to a carrier molecule arewell known in the art (see, for example, Harlow and Lane, supra, 1988).An immunogenic Bcl-G polypeptide fragment can also be generated byexpressing the peptide portion as a fusion protein, for example, toglutathione S transferase (GST), polyHis or the like. Methods forexpressing peptide fusions are well known to those skilled in the art(Ausubel et al., Current Protocols in Molecular Biology (Supplement 47),John Wiley & Sons, New York (1999)).

The invention further provides a method for detecting the presence of ahuman Bcl-G in a sample by contacting a sample with a Bcl-G-specificantibody, and detecting the presence of specific binding of the antibodyto the sample, thereby detecting the presence of a human Bcl-G in thesample. Bcl-G specific antibodies can be used in diagnostic methods andsystems to detect the level of Bcl-G present in a sample. As usedherein, the term “sample” is intended to mean any biological fluid,cell, tissue, organ or portion thereof, that includes or potentiallyincludes Bcl-G nucleic acids or polypeptides. The term includes samplespresent in an individual as well as samples obtained or derived from theindividual. For example, a sample can be a histologic section of aspecimen obtained by biopsy, or cells that are placed in or adapted totissue culture. A sample further can be a subcellular fraction orextract, or a crude or substantially pure nucleic acid or proteinpreparation.

Bcl-G-specific antibodies can also be used for the immunoaffinity oraffinity chromatography purification of the invention Bcl-G. Inaddition, methods are contemplated herein for detecting the presence ofan invention Bcl-G protein in a cell, comprising contacting the cellwith an antibody that specifically binds to Bcl-G polypeptides underconditions permitting binding of the antibody to the Bcl-G polypeptides,detecting the presence of the antibody bound to the Bcl-G polypeptide,and thereby detecting the presence of invention polypeptides in a cell.With respect to the detection of such polypeptides, the antibodies canbe used for in vitro diagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of target Bcl-Gpolypeptides in a sample include immunoassays that employ a detectableantibody. Such immunoassays include, for example, immunohistochemistry,immunofluorescence, ELISA assays, radioimmunoassay, FACS analysis,immunoprecipitation, immunoblot analysis, Pandex microfluorimetricassay, agglutination assays, flow cytometry and serum diagnostic assays,which are well known in the art (Harlow and Lane, supra, 1988; Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborPress (1999)).

An antibody can be made detectable by various means well known in theart. For example, a detectable marker can be directly attached to theantibody or indirectly attached using, for example, a secondary agentthat recognizes the Bcl-G specific antibody. Useful markers include, forexample, radionucleotides, enzymes, binding proteins such as biotin,fluorogens, chromogens and chemiluminescent labels.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturation to form afluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

In addition to detecting the presence of a Bcl-G polypeptide, inventionanti-Bcl-G antibodies are contemplated for use herein to modulate theactivity of the Bcl-G polypeptide in living animals, in humans, or inbiological tissues or fluids isolated therefrom. The term “modulate”refers to a compound=s ability to increase the biological activity byfunctioning as an agonist or inhibit the biological activity byfunctioning as an antagonist of an invention Bcl-G polypeptide.Accordingly, compositions comprising a carrier and an amount of anantibody having specificity for Bcl-G polypeptides effective to blocknaturally occurring ligands or other Bcl-G-binding proteins from bindingto invention Bcl-G polypeptides are contemplated herein. For example, amonoclonal antibody directed to an epitope of an invention Bcl-Gpolypeptide, including an amino acid sequence set forth in SEQ ID NOS:2,4 or 42, can be useful for this purpose.

The present invention further provides transgenic non-human mammals thatare capable of expressing exogenous nucleic acids encoding Bcl-Gpolypeptides. As employed herein, the phrase “exogenous nucleic acid”refers to nucleic acid sequence which is not native to the host, orwhich is present in the host in other than its native environment, forexample, as part of a genetically engineered DNA construct. In additionto naturally occurring levels of Bcl-G, a Bcl-G polypeptide of theinvention can either be overexpressed or underexpressed in transgenicmammals, for example, underexpressed in a knock-out animal.

Also provided are transgenic non-human mammals capable of expressingnucleic acids encoding Bcl-G polypeptides so mutated as to be incapableof normal activity. Therefore, the transgenic non-human mammals do notexpress native Bcl-G or have reduced expression of native Bcl-G. Thepresent invention also provides transgenic non-human mammals having agenome comprising antisense nucleic acids complementary to nucleic acidsencoding Bcl-G polypeptides, placed so as to be transcribed intoantisense mRNA complementary to mRNA encoding Bcl-G polypeptides, whichhybridizes to the mRNA and, thereby, reduces the translation thereof.The nucleic acid can additionally comprise an inducible promoter and/ortissue specific regulatory elements, so that expression can be induced,or restricted to specific cell types.

Animal model systems useful for elucidating the physiological andbehavioral roles of Bcl-G polypeptides are also provided, and areproduced by creating transgenic animals in which the expression of theBcl-G polypeptide is altered using a variety of techniques. Examples ofsuch techniques include the insertion of normal or mutant versions ofnucleic acids encoding an Bcl-G polypeptide by microinjection,retroviral infection or other means well known to those skilled in theart, into appropriate fertilized embryos to produce a transgenic animal,see, for example, Hogan et al., Manipulating the Mouse Embryo: ALaboratory Manual (Cold Spring Harbor Laboratory, (1986)). Transgenicanimal model systems are useful for in vivo screening of compounds foridentification of specific ligands, such as agonists or antagonists,which activate or inhibit a biological activity.

Also contemplated herein, is the use of homologous recombination ofmutant or normal versions of Bcl-G genes with the native gene locus intransgenic animals, to alter the regulation of expression or thestructure of Bcl-G polypeptides by replacing the endogeneous gene with arecombinant or mutated Bcl-G gene. Methods for producing a transgenicnon-human mammal including a gene knock-out non-human mammal, are wellknown to those skilled in the art (see, Capecchi et al., Science244:1288 (1989); Zimmer et al., Nature 338:150 (1989); Shastry,Experentia, 51:1028-1039 (1995); Shastry, Mol. Cell. Biochem.,181:163-179 (1998); and U.S. Pat. No. 5,616,491, issued Apr. 1, 1997,No. 5,750,826, issued May 12, 1998, and No. 5,981,830, issued Nov. 9,1999).

Invention nucleic acids, oligonucleotides, including antisense, vectorscontaining invention nucleic acids, transformed host cells, polypeptidesand combinations thereof, as well as antibodies of the presentinvention, can be used to screen compounds to determine whether acompound functions as a potential agonist or antagonist of inventionpolypeptides. These screening assays provide information regarding thefunction and activity of invention polypeptides, which can lead to theidentification and design of compounds that are capable of specificinteraction with one or more types of polypeptides, peptides orproteins.

Thus, the invention provides methods for identifying compounds whichbind to Bcl-G polypeptides. The invention proteins can be employed in acompetitive binding assay. Such an assay can accommodate the rapidscreening of a large number of compounds to determine which compounds,if any, are capable of binding to Bcl-G polypeptides. Subsequently, moredetailed assays can be carried out with those compounds found to bind,to further determine whether such compounds act as modulators, agonistsor antagonists of invention Bcl-G polypeptides. Compounds that bind toand/or modulate invention Bcl-G polypeptides can be used to treat avariety of pathologies mediated by invention Bcl-G polypeptides.

Various binding assays to identify cellular proteins that interact withprotein binding domains are known in the art and include, for example,yeast two-hybrid screening assays (see, for example, U.S. Pat. Nos.5,283,173, 5,468,614 and 5,667,973; Ausubel et al., supra, 1999; Lubanet al., Curr. Opin. Biotechnol. 6:59-64 (1995)) and affinity columnchromatography methods using cellular extracts. By synthesizing orexpressing polypeptide fragments containing various Bcl-G sequences ordeletions, the Bcl-G binding interface can be readily identified.

In another embodiment of the invention, there is provided a bioassay foridentifying compounds which modulate the activity of invention Bcl-Gpolypeptides. According to this method, invention polypeptides arecontacted with an “unknown” or test substance, for example, in thepresence of a reporter gene construct responsive to a Bcl-G signalingpathway, the activity of the polypeptide is monitored subsequent to thecontact with the “unknown” or test substance, and those substances whichcause the reporter gene construct to be expressed are identified asfunctional ligands for Bcl-G polypeptides. Such reporter gene assays andsystems are well known to those skilled in the art (Ausubel et al.,supra, 1999). In addition, a reporter gene constrict can be generatedusing the promoter region of Bcl-G and screened for compounds thatincrease or decrease Bcl-G gene promoter activity. Such compounds canalso be used to alter Bcl-G expression.

In accordance with another embodiment of the present invention,transformed host cells that recombinantly express invention polypeptidescan be contacted with a test compound, and the modulating effect(s)thereof can then be evaluated by comparing the Bcl-G-mediated response,for example, via reporter gene expression in the presence and absence oftest compound, or by comparing the response of test cells or controlcells, to the presence of the compound.

As used herein, a compound or a signal that “modulates the activity” ofinvention polypeptides refers to a compound or a signal that alters theactivity of Bcl-G polypeptides so that the activity of the inventionpolypeptide is different in the presence of the compound or signal thanin the absence of the compound or signal. In particular, such compoundsor signals include agonists and antagonists. An agonist encompasses acompound or a signal that activates Bcl-G protein expression orbiological activity. Alternatively, an antagonist includes a compound orsignal that interferes with Bcl-G expression or biological activity.Typically, the effect of an antagonist is observed as a blocking ofagonist-induced protein activation. Antagonists include competitive andnon-competitive antagonists.

Assays to identify compounds that modulate Bcl-G polypeptide expressioncan involve detecting a change in Bcl-G polypeptide abundance inresponse to contacting the cell with a compound that modulates Bcl-Gactivity. Assays for detecting changes in polypeptide expressioninclude, for example, immunoassays with Bcl-G-specific Bcl-G antibodies,such as immunoblotting, immunofluorescence, immunohistochemistry andimmunoprecipitation assays, as described above.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate Bcl-G activity generally requirecomparison to a control. One type of a “control” is a cell or culturethat is treated substantially the same as the test cell or test cultureexposed to the compound, with the distinction that the “control” cell orculture is not exposed to the compound. Another type of “control” cellor culture can be a cell or culture that is identical to the test cells,with the exception that the “control” cells or culture do not express aBcl-G polypeptide. Accordingly, the response of the transfected cell toa compound is compared to the response, or lack thereof, of the“control” cell or culture to the same compound under the same reactionconditions.

Methods for producing pluralities of compounds to use in screening forcompounds that modulate the activity of a Bcl-G polypeptide, includingchemical or biological molecules such as simple or complex organicmolecules, metal-containing compounds, carbohydrates, peptides,proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids,antibodies, and the like, are well known in the art and are described,for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr.Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol.,2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al.,Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containinglarge numbers of natural and synthetic compounds also can be obtainedfrom commercial sources. Combinatorial libraries of molecules can beprepared using well known combinatorial chemistry methods (Gordon etal., J. Med. Chem. 37: 1233-1251 (1994); Gordon et al., J. Med. Chem.37: 1385-1401 (1994); Gordon et al., Acc. Chem. Res. 29:144-154 (1996);Wilson and Czarnik, eds., Combinatorial Chemistry: Synthesis andApplication, John Wiley & Sons, New York (1997)).

Compounds that modulate Bcl-G activity can be screened by the methodsdisclosed herein to identify compounds that modulate any biologicalactivity or function of Bcl-G. For example, compounds can be identifiedthat alter the interaction of Bcl-G with Bcl-2 family members.Additionally, compounds can be identified that modulate ion channelactivity associated with Bcl-G. The formation of ion channels by Bcl-2family members is one mechanism of inducing apoptosis in cells (Reed,supra, 1998). Therefore, compounds that modulate ion channel activity ofBcl-G can be used to alter apoptosis, thereby increasing or decreasingapoptotic activity of Bcl-G.

Another assay for screening of compounds that modulates the activity ofBcl-G is based on altering the phenotype of yeast by expressing Bcl-G.For example, expression of Bax in yeast confers a lethal phenotype(Matsuyama et al., Mol. Cell. 1:327-336 (1998)). A yeast that expressesBcl-G can have a similar phenotype as Bax since the biological activityof Bcl-G is similar to Bax (Example III). Accordingly, a yeast strainexpressing Bcl-G that confers a lethal phenotype can be screened forcompounds that prevent cell death. In one embodiment, expression ofBcl-G can be inducible (Tao et al., J. Biol. Chem. 273:23704-23708(1998), and the compounds can be screened when Bcl-G expression isinduced. Bcl-G can also be co-expressed in yeast with other Bcl-2 familymembers having anti-apoptotic activity such as Bcl-2 or Bcl-X_(L). Forexample, co-expression of Bax with Bcl-2 or Bcl-X_(L) suppressed thelethal activity of Bax (Tao et al., supra, 1998). Similarly,co-expression of Bcl-G with an anti-apoptotic Bcl-2 family member suchas Bcl-2 or Bcl-X_(L) can be used to screen for compounds thatantagonize the activity of the anti-apoptotic Bcl-2 family members andrestore a lethal phenotype. Such compounds can function to inhibitbinding of Bcl-G to anti-apoptotic Bcl-2 family members such as Bcl-2 orBcl-X_(L).

In yet another embodiment of the present invention, the activation ofBcl-G polypeptides can be modulated by contacting the polypeptides withan effective amount of at least one compound identified by the assaysdescribed herein. The invention also provides a method of identifying aneffective agent that alters the association of a Bcl-G with a Bcl-Gassociated polypeptide (BAP). The method includes the steps ofcontacting the Bcl-G and the BAP polypeptide, under conditions thatallow said Bcl-G and BAP polypeptide to associate, with a compound; anddetecting the altered association of the Bcl-G and BAP polypeptide,thereby identifying a compound that is an effective agent for alteringthe association of Bcl-G with BAP. The compound can be, for example, adrug or polypeptide. A BAP can be, for example, Bcl-2 family member suchas Bcl-2 or Bcl-X_(L).

As disclosed herein, Bcl-G is a new member of the Bcl-2 family that haspro-apoptotic activity (see Example III). Therefore, modulation of Bcl-Gactivity can be advantageously used to modulate the level of apoptosisin a cell. For example, increasing the activity of Bcl-G can be used topromote apoptosis in a cell. Bcl-G activity can be increased, forexample, by increasing the level of a Bcl-G polypeptide or functionalfragment thereof. Increased levels of a Bcl-G polypeptide can beaccomplished, for example, by delivering to a cell a nucleic acidencoding Bcl-G and expressing a Bcl-G polypeptide recombinantly or bydelivering a Bcl-G polypeptide or functional fragment thereof directlyto a target by the methods disclosed herein. Additionally, Bcl-Gactivity can be increased by using a modulatory agent that functions asan agonist. Promoting apoptosis by increasing Bcl-G activity orexpression is useful, for example, in therapeutic applications such asthe treatment of cancer.

As disclosed herein, decreases or loss of Bcl-G is associated withapproximately 50% of prostate cancers, approximately 30% of ovariancancers and approximately 30% of leukemias. Bcl-G can function as atumor suppressor. Therefore, methods of administering a Bcl-Gpolypeptide either directly or using an encoded nucleic acid can be usedto treat a cancer. Furthermore, many chemotherapeutic agents functionthrough increasing apoptosis. Therefore, the invention additionallyprovides a method to enhance a chemotherapy by increasing Bcl-G activityor expression. Administering Bcl-G can thus be used to enhance theeffect of standard chemotherapeutic agents.

Alternatively, modulation of Bcl-G activity can be advantageously usedto decrease Bcl-G activity to decrease apoptosis. For example, Bcl-Gactivity or expression can be decreased by administering an anti-senseBcl-G nucleic acid. In addition, an antagonist of Bcl-G activity can beidentified by the methods disclosed herein and used to decrease Bcl-Gactivity. Decreasing Bcl-G activity can be used to inhibit apoptosisInhibiting apoptosis can be useful, for example, to treat diseaseischemic. For example, decreasing Bcl-G activity with anti-sense nucleicacids or small molecule compounds can be used to treate stroke, heartattack, autoimmunity, trauma, neuron cell death, and inflammatorydiseases, including Crohn's disease. For example, Bcl-G was identifiedin Crohn's disease patients (see Example I).

The invention further provides a method for modulating an activitymediated by a Bcl-G polypeptide by contacting the Bcl-G polypeptide withan effective, modulating amount of an agent that modulates Bcl-Gactivity. The Bcl-G activity can be, for example, apoptosis-inducingactivity, binding to Bcl-2, or tumor suppressor activity. The inventionadditionally provides a method of modulating the level of apoptosis in acell. The method includes the steps of introducing a nucleic acidmolecule encoding a Bcl-G into the cell; and expressing the Bcl-G in thecell, wherein the expression of the Bcl-G modulates apoptosis in thecell.

The invention further provides a method of modulating the level ofapoptosis in a cell by contacting the cell with a compound thateffectively alters the association of Bcl-G with aBcl-G-associated-protein in the cell, or that effectively alters theactivity of a Bcl-G in the cell. Additionally provided by the inventionis a method of modulating interactions between Bcl-G and Bcl-2 bycontacting a Bcl-G polypeptide with the agent that inhibits or altersinteractions between Bcl-G and Bcl-2.

As disclosed herein, Bcl-G is located on chromosome 12 in a regiondeleted in various cancers, including leukemia, prostate and ovariancancer (Example IV). Therefore, methods using Bcl-G nucleic acids orantibodies can be used as a diagnostic for predisposition or progressionof cancer, for example, leukemia, prostate or ovarian cancer. Changes inBcl-G expression or activity can be correlated with patient survival orresponse to therapy, and a correlation can be used to monitor cancerprogression or response to therapy.

The invention further provides a method of diagnosing a pathologycharacterized by an increased or decreased level of a Bcl-G in asubject. The method includes the steps of (a) obtaining a test samplefrom the subject; (b) contacting the sample with an agent that can bindthe Bcl-G under suitable conditions, wherein the conditions allowspecific binding of the agent to the Bcl-G; and (c) comparing the amountof the specific binding in the test sample with the amount of specificbinding in a control sample, wherein an increased or decreased amount ofthe specific binding in the test sample as compared to the controlsample is diagnostic of a pathology. The agent can be, for example, ananti-Bcl-G antibody, a Bcl-G-associated-protein (BAP), or a Bcl-Gnucleic acid.

The invention also provides a method of diagnosing cancer or monitoringcancer therapy by contacting a test sample from a patient with aBcl-G-specific antibody. The invention additionally provides a method ofassessing prognosis of patients with cancer comprising contacting a testsample from a patient with a Bcl-G-specific antibody.

The invention additionally provides a method of diagnosing cancer ormonitoring cancer therapy by contacting a test sample from a patientwith a Bcl-G oligonucleotide. The invention further provides a method ofassessing prognosis of patients with cancer by contacting a test samplefrom a patient with a Bcl-G oligonucleotide.

The methods of the invention for diagnosing cancer or monitoring cancertherapy using a Bcl-G-specific antibody or Bcl-G oligonucleotide ornucleic acid can be used, for example, to segregate patients into a highrisk group or a low risk group for predicting risk of metastasis or riskof failure to respond to therapy. Therefore, the methods of theinvention can be advantageously used to determine the risk of metastasisin a cancer patient or as a prognostic indicator of survival in a cancerpatient. One of ordinary skill in the art would appreciate that theprognostic indicators of survival for cancer patients suffering fromstage I cancer can be different from those for cancer patients sufferingfrom stage IV cancer. For example, prognosis for stage I cancer patientscan be oriented toward the likelihood of continued growth and/ormetastasis of the cancer, whereas prognosis for stage IV cancer patientscan be oriented toward the likely effectiveness of therapeutic methodsfor treating the cancer. Accordingly, the methods of the inventiondirected to measuring the level of or determining the presence of aBcl-G polypeptide or encoding nucleic acid can be used advantageously asa prognostic indicator for the presence or progression of a cancer orresponse to therapy.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems, preferably in kit form, comprising atleast one invention nucleic acid or antibody in a suitable packagingmaterial. The diagnostic kits containing nucleic acids are derived fromthe Bcl-G-encoding nucleic acids described herein. In one embodiment,for example, the diagnostic nucleic acids are derived from any of SEQ IDNOS:1, 3 or 41 and can be oligonucleotides of the invention. Inventiondiagnostic systems are useful for assaying for the presence or absenceof nucleic acid encoding Bcl-G in either genomic DNA or mRNA.

A suitable diagnostic system includes at least one invention nucleicacid or antibody, as a separately packaged chemical reagent(s) in anamount sufficient for at least one assay. For a diagnostic kitcontaining nucleic acid of the invention, the kit will generally containtwo or more nucleic acids. When the diagnostic kit is to be used in PCR,the kit will contain at least two oligonucleotides that can serve asprimers for PCR. Those of skill in the art can readily incorporateinvention nucleic probes and/or primers or invention antibodies into kitform in combination with appropriate buffers and solutions for thepractice of the invention methods as described herein. A kit containinga Bcl-G antibody can contain a reaction cocktail that provides theproper conditions for performing an assay, for example, an ELISA orother immunoassay, for determining the level of expression of a Bcl-Gpolypeptide in a sample, and can contain control samples that containknown amounts of a Bcl-G polypeptide and, if desired, a second antibodyspecific for the anti-Bcl-G antibody.

The contents of the kit of the invention, for example, Bcl-G nucleicacids or antibodies, are contained in packaging material, preferably toprovide a sterile, contaminant-free environment. In addition, thepackaging material contains instructions indicating how the materialswithin the kit can be employed both to detect the presence or absence ofa particular Bcl-G sequence or Bcl-G polypeptide or to diagnose thepresence of, or a predisposition for a condition associated with thepresence or absence of Bcl-G such as cancer. The instructions for usetypically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Molecular Cloning of Bcl-G

This example describes the cloning of Bcl-G, a homologue of Bcl-2.

To clone the full length Bcl-G gene, oligonucleotide primers weredesigned based a short EST (GenBank Accession No. AW000827) from colonicmucosa of 3 patients with Crohn's disease found by searching a databasefor sequences similar to the BH2 and BH3 domains of Bcl-2 familyproteins. The primers used were Primer 1 (5′GTACTTGGTGCCAAAGCCCAGG-3;SEQ ID NO:7) and Primer 2 (5′-GACATGATGTCTGGTGTAGTAGGCGAGG-3; SEQ DINO:8). The full length Bcl-G cDNA was cloned using SMART™RACE cDNAAmplification Kit (Clontech; Palo Alto Calif.) from human placentaltotal RNA (Clontech) as template. The 5′-RACE products were sequencedwith an automated sequencer.

Briefly, for cloning of Bcl-G cDNAs, TBLAST searches of the publicdatabases using human Bcl-2 as a query sequence revealed a short EST(GenBank AW000827) from colonic mucosa of 3 patients with Crohn=sdisease which contains an open reading frame (ORF) encoding sequencessimilar to the BH2 domain of Bcl-2 family proteins. An oligonucleotideprimer (5=-GTACTTGGTGCCAAAGCCCAGG-3=; SEQ ID NO:7) was designedcomplementary to the EST sequence and used for 5=-RACE, employing theSMART

RACE cDNA Amplification Kit (Clontech; Palo Alto Calif.) and humanplacental total RNA as template. The 5=-RACE products were subclonedinto pCR2.1-TOPO vector using the TOPO

TA Cloning kit (Invitrogen; Carlsbad Calif.), and their DNA sequencedetermined, revealing a complete open reading frame (ORF), with startcodon within a favorable Kozak sequence context, preceded by a5=-untranslated region (UTR) containing stop codons in all threereading-frames (submitted to Genbank). Two additional EST clones,AI478889 and AI240211, were identified by BLAST searches, correspondingto overlapping partial Bcl-G cDNAs which contained the 3′ BUTR.

A short EST was identified during searches of the public databases,which when conceptually translated revealed a polypeptide sequence withsimilarity to the BH2 domain of Bcl-2 family proteins. Full-length cDNAswere obtained, revealing two potential transcripts containing openreading frames (ORF) for proteins of 327 and 252 amino-acids,respectively, which were termed Bcl-G_(L) and Bcl-G_(S) (FIG. 5A). Thepredicted Bcl-G_(L) and Bcl-G_(S) proteins are identical for the first226 amino acids, then diverge thereafter. Comparison of the predictedamino acid sequences of Bcl-G_(L) and Bcl-G_(S) with Bcl-2 familyproteins revealed the presence of a candidate BH3 domain (SEQ ID NO:9)in both Bcl-G_(L) and Bcl-G_(S) (FIG. 5A,B), and the presence of a BH2domain (SEQ ID NO:18) in Bcl-G_(L) but not in Bcl-G_(S) (FIG. 5A, C).

Invention Bcl-G was found to exist in two forms, a long form, designatedBcl-G_(L), and a shorter form, designated Bcl-G_(S). The nucleotidesequence of Bcl-G_(L) is shown in FIG. 1 (SEQ ID NO:1). The nucleotidesequence of the coding region of Bcl-G_(L) cDNA and the encoded aminoacid sequence (SEQ ID NO:3) are shown in FIG. 2. Bcl-G_(L) was initiallyidentified to contain a core BH3 domain (²¹⁶LKYSGDQLE²²⁴; SEQ ID NO:5)and a core BH2 domain (³⁰⁷PWIQQHGGWE³¹⁶; SEQ ID NO:6).

The shorter form of Bcl-G, Bcl-G_(S), is an apparent alternativesplicing product of Bcl-G mRNA. The nucleotide sequence of Bcl-G_(S) isshown in FIG. 3 (SEQ ID NO:3). The nucleotide sequence of the codingregion of Bcl-G_(S) cDNA and the encoded amino acid sequence (SEQ IDNO:4) are shown in FIG. 2. Bcl-G_(S) contains only the BH3 domain(²¹⁶LKYSGDQLE²²⁴).

These results demonstrate that a new member of the Bcl-2 family, Bcl-G,is expressed in human placenta and in the colonic mucosa of patientswith Crohn's disease. Bcl-G exists in two forms, a long form, designatedBcl-G_(L), which contains a BH2 and BH3 domain, and a short form,designated Bcl-G_(S), which contains only a BH3 domain.

Example II Mapping of Bcl-G to Chromosome 12p12.3

This experiment describes chromosomal mapping of human Bcl-G.

To map the chromosomal location of Bcl-G, a search of the GenBankdatabase was performed using BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402(1997). A 190858 by human 12p12 BAC chromosome sequence RPCI11-267J23(GenBank accession no. AC007537) was found to contain the full lengthBcl-G gene. The BAC also contains the LRP6 gene (exon 1 starts at 89963bp). A 600 kb region between 12p12.3 to 12p13.1, flanked by D125358 andETV6/exon8, was previously defined to be frequently deleted in childhoodacute lymphoblastic leukemia (ALL) and other solid tumor cells (Baens etal., (1999) Genomics 56:40-50 (1999); Hatta et al., Br. J. Cancer75:1256-1262 (1997); Kibel et al., Cancer Res. 58:5652-5655 (1998);Baccichet et al., Br. J. Haematol. 99:107-114 (1997); Aissani et al.,Leuk. Lymphoma 34:231-239). The loss of the region containing Bcl-Goccurs in approximately 50% of prostate cancers, 30% of ovarian cancers,and 30% of leukemias.

The LRP6 gene is located in the region between 12p12.3 to 12p13.1. UsingLRP6 as a marker for orientation, Bcl-G was located in this region. Exon1 of Bcl-G starts at 40674 by in the BAC and was deduced from novel DNAsequence data obtained from 5′ RACE-based amplification of thefull-length Bcl-G cDNA. The genomic structure of the Bcl-G gene is shownin FIG. 5. Bcl-G_(L) has 6 exons, with the first codon non-coding,spreading across a 30 kb region in chromosome 12. Bcl-G_(S) also has 6exons, but a 153 bps sequence is inserted in front of exon 5 andcontains a stop codon. The BH3 domain is located in exon 4 of bothBcl-G_(L) and Bcl-G_(S). The BH2 domain is located in exon 5 ofBcl-G_(L). The Bcl-G_(L) and Bcl-G_(S) cDNAs can be accounted for by analternative mRNA splicing mechanism in which different splice acceptorsites associated with exon 5 are employed, resulting in a change in thedistal reading-frame (FIG. 5D).

The chromosomal mapping of Bcl-G to chromosome 12p12.3 is shown in FIG.6. Bcl-G is located in a 600 kb region that has been previouslydetermined to be frequently deleted in childhood ALL and other solidtumors (Baens et al., supra, 1999). Therefore, Bcl-G is located in aregion deleted in ALL and can function as a tumor suppressor or as amarker for tumor suppressor activity.

Example III Expression of Bcl-G

This example describes the expression of Bcl-G.

For generation of plasmids, cDNAs containing the ORFs of Bcl-G_(L) andBcl-G_(S) without additional flanking sequences were generated by PCRusing human placental cDNA as a template and the following primers:

5=-GGCTCGAGCGATGTGTAGCACCAGTGGGTGTGACC-3=(SEQ ID NO:27), sense for bothBcl-G_(L) and Bcl-G_(S);5=-CCAAGCTTTAAGTCTACTTCTTCATGTGATATCCC-3=(SEQ ID NO:28), antisense forBcl-G_(L); and5=-CCAAGCTTTAAAATGCAGGCCATCAAACC-3=(SEQ ID NO:29), antisense forBcl-G_(S).The resulting PCR products were digested with restriction endonucleasesand subcloned into the Xho I and Hind III sites of pEGFP-C 1 (Clontech).A mutant of Bcl-G_(S) lacking the BH3 domain was created by a two-stepPCR method, using the following primers: primer1,5=-GGCTCGAGCGATGTGTAGCACCAGTGGGTGTGACC-3=(SEQ ID NO:30); primer2,5=-CCGGATCCGGCTAGTATTTGTTCTTCTTCATCTTTC-3=(SEQ ID NO:31); primer3,5=-CCGGATCCGACACTGCCTTCATCCCCATTCCC-3=(SEQ ID NO:32); and primer4,5=-CCAAGCTTTAAAATGCAGGCCATCAAACC-3=(SEQ ID NO:33). The resulting PCRproduct was digested with XhoI/BamHI or with BamHI/HindIII respectively,and ligated into pEGFP-Cl. Site-directed mutagenesis of Bcl-G_(S) wasperformed to generate a L216E substitution mutation using the QuikChange

Site-Directed Mutagenesis Kit (Stratagene) following manufacturer=sprocedure, with pEGFP-Cl/Bcl-G_(S) plasmid as DNA template, and themutagenic primers: 5=-GCCAAAATTGTTGAGCTGGAGAAATATTCAGGAGATCAGTTGG-3=(SEQID NO:34) and

5=-CCAACTGATCTCCTGAATATTTCTCCAGCTCAACAATTTTGGC-3=(SEQ ID NO:35).

For measurements of Bcl-G mRNAs, Bcl-G mRNAs were detected by eitherNorthern blotting or Reverse-Transcriptase-Polymerase Chain Reaction(RT-PCR). For RT-PCR, multiple-tissue cDNA panels (Clontech) containingfirst-strand cDNA generated from 16 different tissues were employed. PCRwas performed according to the manufacturer=s protocol with followingprimers: (a) 5=primer for both Bcl-G_(S) and Bcl-G_(L), corresponding toexon 3,

5=-CTGAGGGTCTCTCCTTCCAGCTCCAAGG-3=(SEQ ID NO:36); (b) 3=primer forBcl-G_(L), corresponding to exon 5,5=-GGCCGTGACGTCTATTACAAGGGCAGCC-3=(SEQ ID NO:37); and 3=primer forBcl-G_(S), corresponding to an alternatively spliced segment of exon 5,5=-CAAGGGAATGGGGATGAAGGCAGTGTC-3=(SEQ ID NO:38). Human G3PDH expressionwas examined by PCR with the following primers:5-TGAAGGTCGGAGTCAACGGATTTGGT-3=(SEQ ID NO:39) (sense); and5=-CATGTGGGCCATGAGGTCCACCAC-3=(SEQ ID NO:40) (antisense).

For tissue-specific expression of Bcl-G_(L) and Bcl-G_(S) mRNAs,Northern blotting demonstrated the presence of ˜2 kbp Bcl-G transcriptsin several normal human tissues, but failed to resolve the mRNAsencoding Bcl-G_(L) and Bcl-G_(S). RT-PCR assays were therefore designedusing primers specific for Bcl-G_(L) and Bcl-G_(S) sequences associatedwith exon 5. Bcl-G_(L) mRNA was clearly detected in lung, pancreas,prostate and testis, with lower levels present in some other tissues(FIG. 7). In contrast, Bcl-G_(S) mRNA was uniquely expressed in testis.RT-PCR amplification of a control mRNA, G3PDH, demonstrated loading ofnearly equivalent amounts of mRNA from each tissue. The amplified bandscorresponding to Bcl-G_(L) and Bcl-G_(S) were excised and sequenced,confirming the validity of the RT-PCR strategy.

Example IV Induction of Cell Death by Bcl-G_(S)

This experiment describes the induction of cell death by Bcl-G_(S) intransfected PC-3 cells.

For cell culture, transfections, and apoptosis assay, 293T and Cos-7cells were cultured in DMEM high glucose media (Irvine Scientific, SantaAna, Calif.) containing 10% fetal bovine serum (FBS). PC-3 cells werecultured with RPMI 1640 media containing 10% FBS. Transfection of cellswas performed using SuperFect (Qiagen, Chatsworth, Calif.). Bothfloating and adherent cells (after trypsinization) were collected 24 hrsafter transfection, fixed, and stained using4=,6-diamidine-2=-phenylindole dihydrochloride (DAPI) for assessingapoptosis based on nuclear fragmentation and chromatin condensation (Xu& Reed, Mol. Cell, 1:337-346 (1998); Zhang et al., Proc. Natl. Acad.Sci. (USA), 97:2597-2602 (2000)).

To characterize a biological function of Bcl-G, a Bcl-G_(S) constructwas generated by cloning Bcl-G_(S) cDNA into pcDNA3.1/Myc/His expressionvector (Invitrogen; Carlsbad Calif.) at the Xho I/Hind III sites. Theauthenticity of the construct was confirmed by DNA sequencing.

For transfection experiments, various vectors were transfected into PC-3cells: control vector pcDNA3.1/Myc/His; pcDNA3.1/Myc/His/Bcl-G_(S)expressing Bcl-G_(S); pRC/CMV/Bcl-2 expressing Bcl-2; and pRC/CMV/Baxexpressing Bax. The vectors were transfected as follows:pcDNA3.1/Myc/His alone; pcDNA3.1/Myc/His/Bcl-G_(S) alone;pcDNA3.1/Myc/His/Bcl-G_(S)+pRC/CMV/Bcl-2; pRC/CMV/Bax alone; orpRC/CMV/Bax+pRC/CMV/Bcl-2. One μg of each vector was combined with 0.2μg pEGFP—N2 (Clontech), and the vectors were transiently transfectedinto PC-3 prostate cancer cells using SuperFect reagent (QIAGEN;Valencia Calif.), following the instructions of the manufacturer. At 24hours after transfection, cells were examined under a fluorescentmicroscope. About 100 green fluorescent protein (GFP) positive (greencolor) cells were counted for each transfection. Cells that weredetached with membrane blebbing and/or apoptotic bodies were recorded asdead cells. Results were averaged from three separate transfections.

As shown in FIG. 8, Bcl-G_(S) induces cell death in PC-3 cells (compare“control” to “Bcl-G_(S)”). The induction of cell death by Bcl-G_(S) wassimilar to Bax, which was used as a positive control based on its knownpro-apoptotic activity (compare “Bcl-G_(S)” to “Bax”). The induction ofcell death by Bcl-G_(S) was completely inhibited when co-transfectedwith the anti-apoptotic Bcl-2 (see “Bcl-G_(S)+Bcl-2”). The inhibition ofBcl-G_(S)-induced cell death by Bcl-2 was similar to that seen with Bax(see “Bax+Bcl-2”).

To assess the effects of Bcl-G_(L) and further assess the effects ofBcl-G_(S) on apoptosis, various cell lines, including Cos7, HEK293T, andPC3, were transiently transfected with plasmids encoding Bcl-G_(L) orBcl-G_(S). For most experiments, Bcl-G_(L) and Bcl-G_(S) were expressedas GFP-fusions so that successfully transfected cells could beconveniently identified (FIG. 9A), but similar results were obtainedwhen Flag-epitope tags were employed instead. Over-expression of theshorter Bcl-G_(S) protein reproducibly induced striking increases in thepercentage of cells undergoing apoptosis, as determined by DAPI staining(FIG. 9) and other methods. In contrast, Bcl-G_(L) was more variable andless efficient at inducing apoptosis in these transient transfectionassays. Immunoblot analysis of lysates from transfected cellsdemonstrated that the less potent effects of Bcl-G_(L) could not beaccounted for by lower levels of protein production (FIG. 9A). Indeed,Bcl-G_(L) protein accumulated to levels ˜10-fold higher in cellscompared to Bcl-G_(S), suggesting that Bcl-G_(S) is a far more potentapoptosis-inducer. Analysis of the same blots with an anti-tubulinantibody confirm loading of essentially equivalent amounts of totalprotein for each sample, thus validating the results. In additionaltransfection experiments, Bcl-G_(L) failed to demonstrate cytoprotectiveactivity in side by side comparisons with Bcl-2 and Bcl-X_(L).

Example V The BH3 Domain of Bcl-G_(S) is Required for its Pro-ApoptoticActivity

The Bcl-G_(S) protein contains a BH3 domain, but lacks other regions ofhomology with Bcl-2 family proteins. Structural studies indicate thatBH3 domains represent amphipathic a-helices, in which the hydrophobicsurface of the a-helices of apoptosis-inducing BH3 peptides bind to apocket on survival proteins such as Bcl-X_(L) (Sattler et al., Science,275:983-986 (1997)). Therefore, the apoptosis-inducing activity of thewild-type Bcl-G_(S) protein was compared with mutants lacking the BH3domain (ΔBH3) or in which leucine 216 within the BH3 domain of Bcl-G_(S)was chosen for mutation to charged glutamic acid, based on comparisonswith previously described BH3 mutagenesis experiments demonstrating acritical requirement for the analogous leucine in other pro-apoptoticBcl-2 family proteins (Wang et al., Mol. Cell. Biol., 18:6083-6089(1998); Kelekar et al., Mol. Cell. Biol., 17:7040-7046 (1997)).

Wild-type Bcl-G_(S) potently induced apoptosis when overexpressed inCos-7, PC3, HEK293T and other cell lines, whereas Bcl-G_(S) (ΔBH3) andBcl-G_(S) (L216E) did not (see FIG. 3B). Immunblot analysis confirmedproduction of the Bcl-G_(S) (ΔBH3) and Bcl-G_(S) (L216E) proteins atlevels exceeding the amounts of wild-type Bcl-G_(S) protein. Therefore,the BH3 domain of Bcl-G_(S) is critical for its pro-apoptotic activity.

Example VI Bcl-G_(S) Associates with Bcl-X_(L) in a BH3-Dependent Manner

The pro-apoptotic activity of “BH3-only” members of the Bcl-2 familydepends on their ability to dimerize with and suppress the activity ofsurvival proteins such as Bcl-X_(L) (reviewed in Kelekar & Thompson,Trends Cell Biol., 8:324-330 (1998)). It was, therefore, determinedwhether Bcl-G_(L) and Bcl-G_(S) are capable of associating with otherBcl-2 family proteins by co-immunoprecipitation assays.

For co-immunoprecipitations and immunoblotting, immunoblotting wasperformed as described previously (Xu and Reed, supra., (1998); Zhang etal., supra., (2000)). For co-immunoprecipitations, cells were culturedin 50 mM benzocarbonyl Valine Alanine Aspartate fluoromethyl-ketone(zVAD-fmk) to prevent apoptosis. Cells were suspended in lysis buffer(50 mM Tris-HCl, pH7.4; 150 mM NaCl; 20 mM EDTA; 50 mM NaF; 0.5% NP-40;0.1 mM Na₃VO₄; 20 μg/ml Leupeptin; 20 μg/ml Aprotinin; 1 mMdithiothreitol (DTT); and 1 mM phenylmethylsulfonylfluoride (PMSF).Lysates (0.2 ml diluted into 1 ml final volume of lysis buffer) werecleared by incubation with 15 μl of protein G-Sepharose 4B (Zymed; SouthSan Francisco Calif.) and then incubated with 15 μl of polyclonalanti-GFP antibody (Santa Cruz; Santa Cruz Calif.) and 15 μl of protein Gat 4EC overnight. Beads were then washed 4 times with 1.5 mls lysisbuffer before boiling in Laemmli sample buffer and performingSDS-PAGE/immunoblotting.

Bcl-Gs association with the survival proteins Bcl-X_(L) and Bcl-2 wasreadily detected by co-immunoprecipitation using lysates fromtransiently transfected cells, whereas no association with pro-apoptoticproteins Bax, Bak, Bid or Bad was observed (FIG. 10A). Interaction ofBcl-G_(S) with Bcl-2 and Bcl-X_(L), but not with Bax or Bak, was alsoconfirmed by yeast two-hybrid assays. In contrast, association of thelonger Bcl-G_(L) protein with Bcl-2 or Bcl-X_(L) was not easily detectedby co-immunoprecipitation assays (FIG. 10A). With much longer x-ray filmexposure times, however, small amounts of Bcl-X_(L) were observed inassociation with Bcl-G_(L) immunocomplexes, suggesting either lowaffinity binding of Bcl-G_(L) to Bcl-X_(L) or implying that only a smallportion of total Bcl-G_(L) proteins are competent to bind Bcl-X_(L). Theinteraction of Bcl-G_(S) with Bcl-X_(L) was BH3-dependent, as determinedby comparisons of wild-type Bcl-G_(S) with the Bcl-G_(S) (ΔBH3) andBcl-G_(S) (L216E) proteins (FIGS. 10B, C). Thus, the pro-apoptoticactivity of Bcl-G_(S) correlates with it ability to bind Bcl-X_(L).

Example VII Bcl-G_(S) is Associated with Cytosolic Organelles

Many Bcl-2 family proteins, such as Bcl-2, Bcl-X_(L), and Bak, contain ahydrophobic stretch of amino-acids near their carboxyl-terminus thatanchors them in intracellular membranes of mitochondria, endoplasmicreticulum, or nuclear envelope (reviewed in Reed, J. C., Nature,387:773-776 (1997); Adams & Cory, Science, 281:1322-1326 (1998); Grosset al., Genes Dev., 13:1899-1911 (1999)). However, some pro-apoptoticBcl-2 family proteins, such as Bax, Bid, and Bim, are found in thecytosol and must be induced to translocate to mitochondria and otherorganelles where the Bcl-2-family proteins to which they dimerize reside(Wolter et al., J. Cell Biol., 139:1281-1292 (1997); Puthalakath et al.,Mol. Cell, 3:287-96 (1999); Li et al., Cell, 94:491-501 (1998); Luo etal., Cell, 94:481-490 (1998)).

The intracellular locations of the Bcl-G_(L) and Bcl-G_(S) protein wasexamined by confocal microscopy analysis of cells expressing GFP-taggedproteins. GFP-expressing cells were imaged by confocal microscopy usinga Bio-Rad MRC 1024 instrument (Xu & Reed, supra. (1998); Zhang et al.,supra. (2000); Zha et al., Mol. Cell. Biol., 16:6494-6508 (1996)).GFP-Bcl-G_(L) protein was located diffusely throughout cells, similar toGFP control protein (FIG. 11A, B). In contrast, Bcl-G_(S) was found in apunctate cytosolic pattern (FIG. 11C), suggestive of organelleassociation. Surprisingly, deletion of the BH3 domain from Bcl-G_(S) didnot disrupt the punctate distribution (FIG. 5D), indicating that otherregions of the Bcl-G_(S) protein are sufficient for subcellulartargeting. Subcellular fractionation experiments confirmed theseobservations, demonstrating association of Bcl-G_(S) and Bcl-G_(S)(DB43) predominantly with organelle-containing heavy-membrane fractions,with scant amounts in the soluble cytosolic compartment.

Example VIII Loss of Heterozygosity (LOH) is Associated with Bcl-G inOvarian Tissue

This example describes loss of heterozygosity (LOH) associated withBcl-G in ovarian cancer tissue.

Ovarian cancer tissue samples were tested for SSCP for possiblemutations in Bcl-G. No mutation was found in exon 1. However, about onethird of the ovarian samples showed a possible LOH of Bcl-G. The LOH wasobserved as a change in band intensity using SSCP. The results wereconfirmed independently using PCR. The LOH samples are sequenced todetermine specific mutations.

These results indicate that LOH is associated with Bcl-G in ovariantissue and can be useful as a marker for ovarian cancer.

Example IX Cloning of Mouse Bcl-G

This example describes cloning of mouse Bcl-G.

The mouse Bcl-G was identified by searching GenBank. An EST clone(AA536718) was found to contain mouse Bcl-G. The EST was purchased fromthe American Type Culture Collection (ATCC; Manassas Va.) and sequencedto determine the complete sequence of mouse Bcl-G.

The nucleotide sequence of mouse Bcl-G cDNA is referenced as SEQ IDNO:41. The amino acid sequence of Bcl-G is referenced as SEQ ID NO:42.

PCR was used to isolate mouse Bcl-G from the purchased EST clone andclone it into EGFP-Cl vector. The primers used were

MXSTA, (SEQ ID NO: 43) 5′-GGGCTCGAGATGTGCAGCACCAGTGTGTATG-3′; NHREV,(SEQ ID NO: 44) 5′-CCAAGCTTTAAGTCTACTTCTTCATGTGATATCCC-3′.

In preliminary experiments, mouse Bcl-G was overexpressed in Cos-7 and293T cells. In these preliminary experiments, apoptosis was notobserved.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

What is claimed is:
 1. A method of identifying an effective agent thatalters the association of a Bcl-G with a Bcl-G associated polypeptide(BAP), comprising the steps of: (a) contacting said Bcl-G and said BAPpolypeptide, under conditions that allow said Bcl-G and BAP polypeptideto associate, with a compound; and (b) detecting the altered associationof said Bcl-G and BAP polypeptide, thereby identifying a compound thatis an effective agent for altering the association of said Bcl-G withBAP.
 2. The method of claim 1, wherein said compound is a compound. 3.The method of claim 1, wherein said compound is a drug.
 4. The method ofclaim 1, wherein said compound is a polypeptide.
 5. A method formodulating an activity mediated by a Bcl-G polypeptide, comprisingcontacting said Bcl-G polypeptide with an effective, modulating amountof an agent identified by claim
 1. 6. The method of claim 5, whereinsaid modulated activity is the binding of Bcl-G to a Bcl-2 familymember.
 7. A therapeutic composition comprising a pharmaceuticallyacceptable carrier and a compound selected from the group consisting ofa Bcl-G polypeptide, a functional fragment of said Bcl-G, a Bcl-Gmodulating compound identified according to claim 1, and an anti-Bcl-Gantibody.
 8. A method of treating a pathology characterized by abnormalcell proliferation, comprising administering an effective amount of thecomposition according to claim
 7. 9. A method of modulating the level ofapoptosis in a cell, comprising contacting the cell with a compound thateffectively alters the association of Bcl-G with aBcl-G-associated-protein in the cell, or that effectively alters theactivity of a Bcl-G in the cell.
 10. A chimeric protein comprising adomain selected from the group consisting of BH3 (SEQ ID NOS:5 or 9) andBH2 (SEQ ID NOS:6 or 18).
 11. A method of modulating interactionsbetween Bcl-G and Bcl-2, comprising contacting a Bcl-G polypeptide withthe agent of claim 1.